Antisense modulation of phosphodiesterase 4D expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of phosphodiesterase 4D. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding phosphodiesterase 4D. Methods of using these compounds for modulation of phosphodiesterase 4D expression and for treatment of diseases associated with expression of phosphodiesterase 4D are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of phosphodiesterase 4D. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding phosphodiesterase 4D. Such compounds have been shown to modulate the expression of phosphodiesterase 4D.

BACKGROUND OF THE INVENTION

[0002] Cyclic nucleotides function as intracellular second messengers in transduction of a variety of extracellular signals. Cyclic nucleotide phosphodiesterases, which degrade cyclic nucleotides to their corresponding monophosphates, play a role in these processes by regulating the intracellular concentration of cyclic nucleotides.

[0003] The phosphodiesterases are represented by a large superfamily of enzymes that possess a modular architecture, with a conserved catalytic domain near the carboxyl terminus and regulatory domains or motifs near the amino terminus. The phosphodiesterase superfamily currently includes 19 different genes sub-grouped into 10 different phosphodiesterase families. Each family is distinguished functionally by its unique combination of enzymatic characteristics (Soderling and Beavo, Curr. Opin. Cell Biol., 2000, 12, 174-179). The existence of multiple phosphodiesterase families, isozymes and splice variants presents an opportunity for complex regulation of cyclic nucleotide levels. Phosphodiesterases 1-7 have been implicated in regulation of insulin secretion, T-cell activation, fertility and growth and erectile function (Soderling and Beavo, Curr. Opin. Cell Biol., 2000, 12, 174-179).

[0004] Phosphodiesterase 4D (also known as cAMP-specific phosphodiesterase 4D, PDE4D, dunce (Drosophila)-homolog phosphodiesterase E3, DPDE3 and rolipram-sensitive 3′,5′-cyclic AMP phosphodiesterase) was first identified as a homolog of the Drosophila dunce gene, one of several genes critical for normal learning and memory in the fly (Milatovich et al., Somat. Cell Mol. Genet., 1994, 20, 75-86). Phosphodiesterase 4D has been localized to chromosome 5q12, a region implicated in spinal muscular atrophy (Milatovich et al., Somat. Cell Mol. Genet., 1994, 20, 75-86).

[0005] Phosphodiesterase 4D was one of 13 phosphodiesterase genes found to be expressed in human cavernous tissue, indicating a potential role in penile erectile regulation and making phosphodiesterase 4D a potential target for the development of drugs for the treatment of erectile dysfunction (Kuthe et al., J. Urol., 2001, 165, 280-283).

[0006] The detection of phosphodiesterase 4 hydrolytic activity and expression of phosphodiesterase 4D in human prostate led Ückert et al. to propose the use of inhibitors of phosphodiesterase 4 enzymes for treating urinary obstruction secondary to benign prostatic hyperplasia (Ückert et al., J. Urol., 2001, 166, 2484-2490).

[0007] Five different proteins produced by alternative splicing of the phosphodiesterase 4D gene (designated PDE4D1, PDE4D2, PDE4D3, PDE4D4 and PDE4D5) were characterized in 1997 (Bolger et al., Biochem. J., 1997, 328, 539-548). When expressed in monkey COS-7 cells, PDE4D1 and PDE4D2 were found to exist only in the cytosol, whereas PDE4D3, PDE4D4 and PDE4D5 were found in both cytosolic and particulate fractions (Bolger et al., Biochem. J., 1997, 328, 539-548). The IC₅₀ values for rolipram, an inhibitor of phosphodiesterase 4 enzymes, were similar for the cytosolic forms of all five variants and 2-7-fold higher for the particulate forms of PDE4D3 and PDE4D5 than their corresponding cytosolic forms. Bolger et al. concluded that the N-terminal regions of PDE4D3, PDE4D4 and PDE4D5 proteins are important in determining their subcellular localization, activity and differential sensitivity to inhibitors (Bolger et al., Biochem. J., 1997, 328, 539-548).

[0008] Miró et al. have identified three additional mRNA variants in human umbilical vein endothelial cells, which code for truncated isoforms of phosphodiesterase 4D designated PDE4DN1, PDE4DN2 and PDE4DN3 and have postulated that these isoforms may participate in the regulation of phosphodiesterase 4D activity (Miró et al., Biochem. Biophys. Res. Commun., 2000, 274, 415-421).

[0009] Erdogan et al. have shown that challenge of human Jurkat T-cells with forskolin leads to induction of the phosphodiesterase 4D variants PDE4D1 and PDE4D2, a finding which may have implications for the use of therapeutic regimens which lead to elevated intracellular cAMP levels and for disease states which involve adenylate cyclase activation (Erdogan and Houslay, Biochem. J., 1997, 321, 165-175).

[0010] Because induction of selective phosphodiesterase 4D subtypes in late pregnancy and labor may influence rates of cAMP hydrolysis in myometrial tissue, Méhats et al. have proposed that a co-application of beta-mimetics and phosphodiesterase 4 inhibitors might improve the tocolytic efficiency of these drugs in the prevention of pre-term delivery (Méhats et al., J. Clin. Endocrinol. Metab., 2001, 86, 5358-5365).

[0011] Nucleic acid sequences encoding phosphodiesterase 4D and isoforms thereof are generally disclosed in U.S. Pat. No. 5,977,305 as well as PCT publications WO 01/79526 and WO 00/23091 (Arkhammar et al., 2000; Terry et al., 2001; Wigler and Colicelli, 1999).

[0012] Disclosed and claimed in PCT publication WO 01/00851 are nucleic acid sequences encoding phosphodiesterase 4D, the complements of said sequences and a process for screening chemical agents for phosphodiesterase modulating activity (Xin et al., 2001).

[0013] To examine the role of phosphodiesterases in cAMP signaling in vivo, Jin et al. inactivated the phosphodiesterase 4D gene in mice. Mice deficient in phosphodiesterase 4D exhibited delayed growth as well as reduced viability and female fertility. The decrease in fertility of the null female was caused by impaired ovulation and diminished sensitivity of the granulosa cells to gonadotropins. These pleiotropic phenotypes demonstrated that phosphodiesterase 4D plays a critical role in cAMP signaling and that its activity is required for the regulation of growth and fertility (Jin et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 11998-12003).

[0014] Muscarinic cholinergic signaling plays an essential role in the control of normal airway functions and in the development of pulmonary disease states, including asthma. Hansen et al (Hansen et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 6751-6756) demonstrated that the airways of mice deficient in phosphodiesterase 4D were no longer responsive to cholinergic stimulation. Airway hyper-reactivity that followed exposure to antigen was also abolished in phosphodiesterase 4D −/− mice, despite apparently normal lung inflammatory infiltration. The loss of cholinergic responsiveness was specific to the airway, not observed in the heart, and was associated with a loss of signaling through muscarinic receptors with an inability to decrease cAMP accumulation. These findings demonstrated that the phosphodiesterase 4D gene plays an essential role in cAMP homeostasis and cholinergic stimulation of the airway, and in the development of hyper-reactivity (Hansen et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 6751-6756).

[0015] Small molecule inhibitors of phosphodiesterase 4D are well known in the art. Examples of phosphodiesterase 4D small molecule inhibitors include rolipram, RS-25344, TVX 2706, quinazolines, napthylridines and thiazoles (Charpiot et al., Bioorg. Med. Chem. Lett., 1998, 8, 2891-2896; Fowler and Odingo, 2001,; Hersperger et al., J. Med. Chem., 2000, 43, 675-682; Saldou et al., Cell. Signal., 1998, 10, 427-440).

[0016] Disclosed and claimed in PCT publication WO 00/23091 are methods for modulation of the effectiveness of phosphodiesterase 4 enzymes and variants thereof, the preferred mode of action being dislocation, disruption of targeting or interference with redistribution of specific isoforms or splice variants of phosphodiesterase 4 (Arkhammar et al., 2000).

[0017] Disclosed and claimed in PCT publication WO 01/79526 are phosphodiesterase 4 dislocators (small molecules, peptides or polypeptides) that remove phosphodiesterase 4 from its native location in the cell (Terry et al., 2001).

[0018] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of phosphodiesterase 4D. To date, investigative strategies aimed at modulating phosphodiesterase 4D expression have involved the use of non-specific small molecule inhibitors and gene knock-outs in mice. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting phosphodiesterase 4D function.

[0019] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of phosphodiesterase 4D.

[0020] The present invention provides compositions and methods for modulating expression of phosphodiesterase 4D, including modulating expression of variants of phosphodiesterase 4D.

SUMMARY OF THE INVENTION

[0021] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding phosphodiesterase 4D, and which modulate the expression of phosphodiesterase 4D. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of phosphodiesterase 4D in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of phosphodiesterase 4D by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding phosphodiesterase 4D, ultimately modulating the amount of phosphodiesterase 4D produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding phosphodiesterase 4D. As used herein, the terms “target nucleic acid” and “nucleic acid encoding phosphodiesterase 4D” encompass DNA encoding phosphodiesterase 4D, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of phosphodiesterase 4D. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0023] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding phosphodiesterase 4D. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding phosphodiesterase 4D, regardless of the sequence(s) of such codons.

[0024] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0025] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0026] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0027] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.

[0028] Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0029] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

[0030] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0031] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0032] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites, including oligonucleotide probes and primers.

[0033] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0034] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0035] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0036] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0037] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the-treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0038] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0039] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0040] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0041] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0042] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0043] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0044] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0045] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0046] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0047] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0048] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C10 alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂ )_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0049] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0050] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0051] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b] [1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b] [1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b] [1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0052] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0053] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0054] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0055] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0056] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0057] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0058] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0059] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0060] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0061] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0062] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0063] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0064] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by-modulating the expression of phosphodiesterase 4D is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0065] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding phosphodiesterase 4D, enabling sandwich and other assays to easily be constructedt to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding phosphodiesterase 4D can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of phosphodiesterase 4D in a sample may also be prepared.

[0066] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0067] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0068] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.

[0069] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0070] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0071] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0072] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0073] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0074] Emulsions

[0075] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0076] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0077] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0078] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0079] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0080] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0081] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0082] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0083] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of-the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0084] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0085] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0086] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0087] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0088] Liposomes

[0089] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0090] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0091] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0092] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0093] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0094] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0095] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0096] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are-ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0097] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0098] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0099] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0100] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0101] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0102] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-demyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0103] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0104] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0105] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0106] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0107] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0108] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0109] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0110] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0111] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0112] Penetration Enhancers

[0113] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0114] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0115] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0116] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0117] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, N.Y., 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0118] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0119] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0120] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0121] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0122] Carriers

[0123] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0124] Excipients

[0125] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0126] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0127] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0128] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0129] Other Components

[0130] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0131] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0132] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0133] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0134] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo-maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0135] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0136] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy amidites

[0137] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.

[0138] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).

[0139] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:

[0140] Preparation of 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite

[0141] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂ were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.

[0142] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite

[0143] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f) 0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0 ° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (R_(f) 0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).

[0144] TLC indicated a complete reaction (product R_(f) 0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.

[0145] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. If desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.

[0146] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite

[0147] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.

[0148] THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA (15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.

[0149] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite)

[0150] 5″-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na₂SO₄), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).

[0151] 2′-Fluoro amidites

[0152] 2′-Fluorodeoxyadenosine amidites

[0153] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′-fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0154] 2′-Fluorodeoxyguanosine

[0155] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyryl-arabinofuranosylguanosine. Alternatively, isobutyryl-arabinofuranosylguanosine was prepared as described by Ross et al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0156] 2′-Fluorouridine

[0157] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0158] 2′-Fluorodeoxycytidine

[0159] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-21-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0160] 2′-0-(2-Methoxyethyl) Modified Amidites

[0161] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0162] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0163] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.

[0164] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at,75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0165] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.

[0166] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate

[0167] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.

[0168] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA (25 mL)) and the fractions were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.

[0169] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite)

[0170] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).

[0171] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0172] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight.

[0173] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3, R_(f) 0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water (3 L) . The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.

[0174] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine Penultimate Intermediate:

[0175] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of >99.7%.

[0176] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′ -O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite)

[0177] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).

[0178] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite)

[0179] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L) . The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of an off-white foam solid (96%).

[0180] Prepartion of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite)

[0181] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).

[0182] 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites

[0183] 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0184] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

[0185] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0186] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×2 00 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.

[0187] 5,-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0188] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution : evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product and R_(f) 0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.

[0189] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0190] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.

[0191] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0192] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH₂Cl₂, and the combined organic phase was washed with water and brine and dried (anhydrous Na₂SO₄). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation.

[0193] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine

[0194] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH₂Cl₂) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation.

[0195] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0196] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH₂Cl₂) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.

[0197] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0198] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation.

[0199] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0200] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P₂O₅ under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0201] 2′-(Aminooxyethoxy) nucleoside amidites

[0202] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

[0203] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0204] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0205] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0206] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-)-O—CH₂—O—CH₂—N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

[0207] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0208] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.

[0209] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine

[0210] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturated NaHCO₃ solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH₂Cl₂/TEA) to afford the product.

[0211] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0212] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0213] Oligonucleotide Synthesis

[0214] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0215] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄oAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0216] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0217] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0218] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0219] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0220] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0221] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0222] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

[0223] Oligonucleoside Synthesis

[0224] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleo-sides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0225] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0226] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0227] PNA Synthesis

[0228] Peptide nucleic acids (PNAS) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

[0229] Synthesis of Chimeric Oligonucleotides

[0230] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0231] [2′-O-Me]—[2′-deoxy]—[2′-0-Ne] Chimeric Phosphorothioate Oligonucleotides

[0232] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligo-nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0233] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0234] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0235] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2N-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0236] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxy phosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0237] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0238] Oligonucleotide Isolation

[0239] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (±32 ±48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0240] Oligonucleotide Synthesis-96 Well Plate Format

[0241] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0242] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0243] Oligonucleotide Analysis—96-Well Plate Format

[0244] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0245] Cell Culture and Oligonucleotide Treatment

[0246] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0247] T-24 Cells:

[0248] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0249] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0250] A549 Cells:

[0251] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0252] NHDF Cells:

[0253] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0254] HEK Cells:

[0255] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0256] Treatment with Antisense Compounds:

[0257] When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0258] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.

Example 10

[0259] Analysis of Oligonucleotide Inhibition of Phosphodiesterase 4D Expression

[0260] Antisense modulation of phosphodiesterase 4D expression can be assayed in a variety of ways known in the art. For example, phosphodiesterase 4D mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0261] Protein levels of phosphodiesterase 4D can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to phosphodiesterase 4D can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997).

[0262] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11

[0263] Poly(A)+ mRNA Isolation

[0264] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HC1, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate-for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0265] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

[0266] Total RNA Isolation

[0267] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0268] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0269] Real-Time Quantitative PCR Analysis of Phosphodiesterase 4D mRNA Levels

[0270] Quantitation of phosphodiesterase 4D mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0271] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0272] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5× PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-well plates containing 30 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0273] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenTM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0274] In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0275] Probes and primers to human phosphodiesterase 4D were designed to hybridize to a human phosphodiesterase 4D sequence, using published sequence information (GenBank accession number U02882.1, incorporated herein as SEQ ID NO:3). For human phosphodiesterase 4D the PCR primers were: forward primer: AGCGCTCAGGAATATCGTAACC (SEQ ID NO: 4) reverse primer: AAAACGCTGTTCGTGAAGATGTC (SEQ ID NO: 5) and the PCR probe was: FAM-TCACCTCCATGTCATCCGAGCA-TAMRA (SEQ ID NO: 6) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:7) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0276] Northern Blot Analysis of Phosphodiesterase 4D mRNA Levels

[0277] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0278] To detect human phosphodiesterase 4D, a human phosphodiesterase 4D specific probe was prepared by PCR using the forward primer AGCGCTCAGGAATATCGTAACC (SEQ ID NO: 4) and the reverse primer AAAACGCTGTTCGTGAAGATGTC (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0279] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0280] Antisense Inhibition of Human Phosphodiesterase 4D Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0281] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human phosphodiesterase 4D RNA, using published sequences (GenBank accession number U02882.1, representing the complete coding sequence of phosphodiesterase 4D, incorporated herein as SEQ ID NO: 3; GenBank accession number AF012073.1, representing the variant PDE4D5, incorporated herein as SEQ ID NO: 10; GenBank accession number AJ250855.1, representing the variant PDE4DN2, incorporated herein as SEQ ID NO: 11; GenBank accession number AU127104.1, representing a variant herein designated PDE4DH1, incorporated herein as SEQ ID NO: 12; GenBank accession number BC008390.1, representing a 5′ extension from the start of SEQ ID NO: 10, incorporated herein as SEQ ID NO: 13, GenBank accession number BG171122.1, representing a 3′ extension from the end of SEQ ID NO: 3, incorporated herein as SEQ ID NO: 14; GenBank accession number L20969.1, representing the variant PDE4D4, incorporated herein as SEQ ID NO: 15; GenBank accession number L20970.1, representing a variant herein designated PDE4DH2, incorporated herein as SEQ ID NO: 16; GenBank accession number U50157.1, representing the variant PDE4D1, incorporated herein as SEQ ID NO: 18; GenBank accession number U50158.1, representing the variant PDE4D2, incorporated herein as SEQ ID NO: 19; and GenBank accession number U50159.1, representing the variant PDE4D3, incorporated herein as SEQ ID NO: 20). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human phosphodiesterase 4D mRNA levels by quantitative real-time PCR as described in otter examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human phosphodiesterase 4D mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO 208721 3′ UTR 3 3862 aagactttattaaggctgac 22 21 208722 3′ UTR 3 3945 ttggcttctatctatctcaa 83 22 208723 3′ UTR 3 4018 ttccacatataaatgctttt 80 23 208724 3′ UTR 3 4147 ataatttttatttcaaagga 9 24 208725 3′ UTR 3 4455 ttaagattctaggcactctg 77 25 208726 3′ UTR 3 4544 cctctctttattagtgcagt 84 26 208727 3′ UTR 3 5374 taaggcacagccctgggacc 38 27 208728 3′ UTR 3 5383 ggttaaagttaaggcacagc 48 28 208729 Start 18 3 atgagggctgctccttcata 69 29 Codon 208730 Coding 18 29 catgctcggatgcccggtgc 86 30 208731 5′ UTR 19 38 tcctcccgccatgctcggat 63 31 208732 Start 20 71 tattcacgtgcatcatgttc 35 32 Codon 208733 Coding 20 85 tctaaagggaaaattattca 0 33 208734 5′ UTR 10 58 aacgtggttccctcgctcac 52 34 208735 Coding 10 367 tcaaaacatgtatgtgccac 46 35 208744 5′ UTR 15 2 aaggctgtatccagggaatt 25 44 208745 5′ UTR 15 139 gctaaataaagtagtctatg 0 45 208746 5′ UTR 15 254 agcctcagggctaccgagag 46 46 208747 5′ UTR 15 357 gctggcccgagagccttcct 46 47 208748 Start 15 414 tctgcctccatcctggctcg 69 48 Codon 208749 Coding 15 793 ggtctccaccgcgtaggagg 16 49 208750 Coding 15 822 ctggatttcttcaggccggg 38 50 208751 Coding 15 854 tgagtccctggaacgaggag 57 51 208752 Coding 15 949 aaaatttgcttggagaatta 15 52 208753 Coding 15 1046 cactggcaatggaggagttc 41 53 208754 Coding 15 1060 atctccgtgtatatcactgg 25 54 208755 Coding 15 1124 caaagttgtttcgtacagtt 7 55 208756 Coding 15 1145 cttgcaaattagttaatgca 16 56 208757 Coding 15 1161 ttgctaggtgctcgatcttg 17 57 208758 Coding 15 1174 catgggtgatcttttgctag 37 58 208759 Coding 15 1203 gtggctttgttgatggatgg 65 59 208760 Coding 15 1301 cggagtgcctggtctgtagg 86 60 208761 Coding 15 1352 tgagctcccgattaagcatc 23 61 208762 Coding 15 1415 agaatgtgtttgatataaac 1 62 208763 Coding 15 1428 tgttgcttatctaagaatgt 0 63 208764 Coding 15 1443 ggaatttccacttcatgttg 15 64 208765 Coding 15 1595 gttccttggcaaggacatct 63 65 208766 Coding 15 1646 acaactctgctattctgaaa 61 66 208767 Coding 15 1730 tatctactggaattttaaat 58 67 208768 Coding 15 1810 atctgcagcatggatattgt 56 68 208769 Coding 15 1868 ctgtaaacacagcctccaaa 55 69 208770 Coding 15 1908 tgtattgcactggcaaaaat 37 70 208771 Coding 15 1913 catcatgtattgcactggca 90 71 208772 Coding 15 1957 gtttgtattgatcagaaatt 32 72 208773 Coding 15 2098 cattttccttaaagattgtc 49 73 208774 Coding 15 2103 atgaccattttccttaaaga 53 74 208775 Coding 15 2119 tgcaagtacgatgtcaatga 1 75 208776 Coding 15 2243 tctgaagaacctgaatccta 41 76 208777 Coding 15 2440 aacaatatagtctatgaagc 35 77 208778 Coding 15 2569 gtcatcaggtgcaggagagg 70 78 208779 Coding 15 2669 cttgactgccactgtccttt 84 79 208780 Stop 15 2841 ttgcactgttacgtgtcagg 88 80 Codon 208781 3′ UTR 15 2909 gttgtggcatgtgacatgca 58 81 208782 3′ UTR 15 3000 tggttacgatattcctgagc 97 82 208783 3′ UTR 15 3087 tgtcagctctaccaagctga 80 83 208784 3′ UTR 15 3168 aaccccaagtccaataaact 33 84 208785 3′ UTR 15 3301 Gctcatcctcctcctactgg 61 85 208786 3′ UTR 15 3525 Cttgcaagttactccttaga 82 86 208787 3′ UTR 15 3804 Tagaacaaatagtcactttg 1 87 208788 5′ UTR 16 49 Agatgaattccaactctctt 7 88 208789 Start 16 113 Tgttcgcagatcttctgtca 0 89 Codon 208790 Coding 16 169 Cacatcaaaacatatccagg 13 90 208791 Coding 13 752 Gtatatgggttcaattccat 21 91 208792 3′ UTR 13 901 Gaatttccaccagtcagcat 40 92 208793 Stop 11 463 Atccttttaaactgttatgg 17 93 Codon 208794 Exon 1c 12 26 Gcacggagcaggaggcactt 62 94 208795 Exon: 12 195 Aaactgtaggactttgagaa 9 95 Exon Junction 208796 Exon: 12 202 Cacatcaaaactgtaggact 2 96 Exon Junction 208797 3′ UTR 14 405 Aagctgatgaatagataacc 72 97 208798 3′ UTR 14 662 Taaatgtttcctgctttagg 88 98

[0282] As shown in Table 1, SEQ ID NOs 22, 23, 25, 26, 28, 29, 30, 31, 34, 35, 46, 47, 48, 51, 59, 60, 65, 66, 67, 68, 69, 71, 73, 74, 78, 79, 80, 81, 82, 83, 85, 86, 94, 97 and 98 demonstrated at least 45% inhibition of human phosphodiesterase 4D expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 16

[0283] Western Blot Analysis of Phosphodiesterase 4D Protein Levels

[0284] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to phosphodiesterase 4D-is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 17

[0285] Targeting of Individual Oligonucleotides to Specific Variants of Phosphodiesterase 4D

[0286] It is advantageous to selectively inhibit the expression of one or more variants of phosphodiesterase 4D. Consequently, in one embodiment of the present invention are oligonucleotides that selectively target, hybridize to, and specifically inhibit one or more, but fewer than all of the variants of phosphodiesterase 4D. A summary of the target sites of the variants is shown in Table 2 and includes GenBank accession number L20973.1, representing PDE4D5, incorporated herein-as SEQ ID NO: 10; GenBank accession number AJ250855.1, representing PDE4DN2, incorporated herein as SEQ ID NO: 11; GenBank accession number AU127104.1, representing PDE4DH1, incorporated herein as SEQ ID NO: 12; GenBank accession number L20969.1, representing PDE4D4, incorporated herein as SEQ ID NO: 15; GenBank accession number L20970.1, representing PDE4DH2, incorporated herein as SEQ ID NO: 16; GenBank accession number U50157.1, representing PDE4D1, incorporated herein as SEQ ID NO: 18; GenBank accession number U50158.1, representing PDE4D2, incorporated herein as SEQ ID NO: 19; GenBank accession number U50159.1, representing PDE4D3, incorporated herein as SEQ ID NO: 20; GenBank accession number AJ250852.1, representing PDE4DN1, incorporated herein as SEQ ID NO: 99; and GenBank accession number AJ250854.1, representing PDE4DN3, incorporated herein as SEQ ID NO: 100. TABLE 2 Targeting of individual oligonucleotides to specific variants of Phosphodiesterase 4D VARIANT OLIGO SEQ SEQ ID ISIS # ID NO. TARGET SITE VARIANT NO. 208729 29 3 PDE4D1 18 208729 29 3 PDE4D2 19 208730 30 29 PDE4D1 18 208730 30 29 PDE4D2 19 208731 31 38 PDE4D2 19 208732 32 71 PDE4D3 20 208732 32 128 PDE4DH2 16 208732 32 15 PDE4DN1 99 208733 33 85 PDE4D3 20 208733 33 142 PDE4DH2 16 208733 33 29 PDE4DN1 99 208734 34 58 PDE4D5 10 208735 35 367 PDE4D5 10 208735 35 250 PDE4DN3 100 208744 44 2 PDE4D4 15 208745 45 139 PDE4D4 15 208746 46 254 PDE4D4 15 208747 47 357 PDE4D4 15 208748 48 414 PDE4D4 15 208749 49 793 PDE4D4 15 208749 49 34 PDE4DN2 11 208750 50 822 PDE4D4 15 208750 50 63 PDE4DN2 11 208751 51 854 PDE4D4 15 208751 51 95 PDE4DN2 11 208752 52 193 PDE4D3 20 208752 52 949 PDE4D4 15 208752 52 452 PDE4D5 10 208752 52 283 PDE4DH1 12 208752 52 250 PDE4DH2 16 208752 52 190 PDE4DN2 11 208752 52 335 PDE4DN3 100 208753 53 290 PDE4D3 20 208753 53 1046 PDE4D4 15 208753 53 549 PDE4D5 10 208753 53 380 PDE4DH1 12 208753 53 347 PDE4DH2 16 208753 53 287 PDE4DN2 11 208753 53 432 PDE4DN3 100 208754 54 304 PDE4D3 20 208754 54 1060 PDE4D4 15 208754 54 563 PDE4D5 10 208754 54 394 PDE4DH1 12 208754 54 361 PDE4DH2 16 208754 54 301 PDE4DN2 11 208754 54 446 PDE4DN3 100 208755 55 368 PDE4D3 20 208755 55 1124 PDE4D4 15 208755 55 627 PDE4D5 10 208755 55 458 PDE4DH1 12 208755 55 425 PDE4DH2 16 208755 55 365 PDE4DN2 11 208755 55 510 PDE4DN3 100 208756 56 1145 PDE4D4 15 208756 56 648 PDE4D5 10 208756 56 479 PDE4DH1 12 208756 56 446 PDE4DH2 16 208756 56 386 PDE4DN2 11 208756 56 531 PDE4DN3 100 208757 57 405 PDE4D3 20 208757 57 1161 PDE4D4 15 208757 57 664 PDE4D5 10 208757 57 495 PDE4DH1 12 208757 57 462 PDE4DH2 16 208757 57 402 PDE4DN2 11 208757 57 547 PDE4DN3 100 208758 58 1174 PDE4D4 15 208758 58 677 PDE4D5 10 208758 58 508 PDE4DH1 12 208758 58 475 PDE4DH2 16 208758 58 415 PDE4DN2 11 208758 58 560 PDE4DN3 100 208759 59 447 PDE4D3 20 208759 59 1203 PDE4D4 15 208759 59 706 PDE4D5 10 208759 59 537 PDE4DH1 12 208759 59 504 PDE4DH2 16 208759 59 444 PDE4DN2 11 208759 59 589 PDE4DN3 100 208760 60 210 PDE4D1 18 208760 60 124 PDE4D2 19 208760 60 545 PDE4D3 20 208760 60 1301 PDE4D4 15 208760 60 136 PDE4DN1 99 208760 60 731 PDE4DN3 100 208761 61 261 PDE4D1 18 208761 61 175 PDE4D2 19 208761 61 596 PDE4D3 20 208761 61 1352 PDE4D4 15 208761 61 855 PDE4D5 10 208761 61 653 PDE4DH2 16 208761 61 480 PDE4DN2 11 208762 62 324 PDE4D1 18 208762 62 238 PDE4D2 19 208762 62 659 PDE4D3 20 208762 62 1415 PDE4D4 15 208762 62 918 PDE4D5 10 208762 62 716 PDE4DH2 16 208762 62 250 PDE4DN1 99 208762 62 845 PDE4DN3 100 208763 63 337 PDE4D1 18 208763 63 251 PDE4D2 19 208763 63 672 PDE4D3 20 208763 63 1428 PDE4D4 15 208763 63 931 PDE4D5 10 208763 63 729 PDE4DH2 16 208763 63 263 PDE4DN1 99 208763 63 858 PDE4DN3 100 208764 61 352 PDE4D1 18 208764 61 266 PDE4D2 19 208764 61 687 PDE4D3 20 208764 61 1443 PDE4D4 15 208764 61 946 PDE4D5 10 208764 61 744 PDE4DH2 16 208764 61 278 PDE4DN1 99 208764 61 873 PDE4DN3 100 208765 65 504 PDE4D1 18 208765 65 418 PDE4D2 19 208765 65 839 PDE4D3 20 208765 65 1595 PDE4D4 15 208765 65 1098 PDE4D5 10 208765 65 896 PDE4DH2 16 208765 65 430 PDE4DN1 99 208765 65 1025 PDE4DN3 100 208766 66 555 PDE4D1 18 208766 66 469 PDE4D2 19 208766 66 890 PDE4D3 20 208766 66 1646 PDE4D4 15 208766 66 1149 PDE4D5 10 208766 66 947 PDE4DH2 16 208766 66 481 PDE4DN1 99 208766 66 1076 PDE4DN3 100 208767 67 639 PDE4D1 18 208767 67 553 PDE4D2 19 208767 67 974 PDE4D3 20 208767 67 1730 PDE4D4 15 208767 67 1233 PDE4D5 10 208767 67 1031 PDE4DH2 16 208767 67 565 PDE4DN1 99 208767 67 1160 PDE4DN3 100 208768 68 719 PDE4D1 18 208768 68 633 PDE4D2 19 208768 68 1054 PDE4D3 20 208768 68 1810 PDE4D4 15 208768 68 1313 PDE4D5 10 208768 69 1111 PDE4DH2 16 208768 68 645 PDE4DN1 99 208768 68 1240 PDE4DN3 100 208769 69 777 PDE4D1 18 208769 69 691 PDE4D2 19 208769 69 1112 PDE4D3 20 208769 69 1868 PDE4D4 15 208769 69 1371 PDE4D5 10 208769 69 1169 PDE4DH2 16 208769 69 703 PDE4DN1 99 208769 69 1298 PDE4DN3 100 208770 70 817 PDE4D1 18 208770 70 731 PDE4D2 19 208770 70 1152 PDE4D3 20 208770 70 1908 PDE4D4 15 208770 70 1411 PDE4D5 10 208770 70 1209 PDE4DH2 16 208770 70 743 PDE4DN1 99 208770 70 1338 PDE4DN3 100 208771 71 822 PDE4D1 18 208771 71 736 PDE4D2 19 208771 71 1157 PDE4D3 20 208771 71 1913 PDE4D4 15 208771 71 1416 PDE4D5 10 208771 71 1214 PDE4DH2 16 208771 71 748 PDE4DN1 99 208771 71 1343 PDE4DN3 100 208772 72 866 PDE4D1 18 208772 72 780 PDE4D2 19 208772 72 1201 PDE4D3 20 208772 72 1957 PDE4D4 15 208772 72 1460 PDE4D5 10 208772 72 1258 PDE4DH2 16 208772 72 792 PDE4DN1 99 208772 72 1387 PDE4DN3 100 208773 73 1007 PDE4D1 18 208773 73 921 PDE4D2 19 208773 73 1342 PDE4D3 20 208773 73 2098 PDE4D4 15 208773 73 1601 PDE4D5 10 208773 73 1399 PDE4DH2 16 208773 73 933 PDE4DN1 99 208773 73 1528 PDE4DN3 100 208774 74 1012 PDE4D1 18 208774 74 926 PDE4D2 19 208774 74 1347 PDE4D3 20 208774 74 2103 PDE4D4 15 208774 74 1606 PDE4D5 10 208774 74 1404 PDE4DH2 16 208774 74 938 PDE4DN1 99 208774 74 1533 PDE4DN3 100 208775 75 1028 PDE4D1 18 208775 75 942 PDE4D2 19 208775 75 1363 PDE4D3 20 208775 75 2119 PDE4D4 15 208775 75 1622 PDE4D5 10 208775 75 1420 PDE4DH2 16 208775 75 954 PDE4DN1 99 208775 75 1549 PDE4DN3 100 208776 76 1152 PDE4D1 18 208776 76 1066 PDE4D2 19 208776 76 1487 PDE4D3 20 208776 76 2243 PDE4D4 15 208776 76 1746 PDE4D5 10 208776 76 1544 PDE4DH2 16 208776 76 1078 PDE4DN1 99 208776 76 1673 PDE4DN3 100 208777 77 1349 PDE4D1 18 208777 77 1263 PDE4D2 19 208777 77 1684 PDE4D3 20 208777 77 2440 PDE4D4 15 208777 77 1943 PDE4D5 10 208777 77 1741 PDE4DH2 16 208777 77 1275 PDE4DN1 99 208777 77 1870 PDE4DN3 100 208778 78 1478 PDE4D1 18 208778 78 1392 PDE4D2 19 208778 78 1813 PDE4D3 20 208778 78 2569 PDE4D4 15 208778 78 2072 PDE4D5 10 208778 78 1870 PDE4DH2 16 208778 78 1404 PDE4DN1 99 208778 78 1999 PDE4DN3 100 208779 79 1578 PDE4D1 18 208779 79 1492 PDE4D2 19 208779 79 1913 PDE4D3 20 208779 79 2669 PDE4D4 15 208779 79 2172 PDE4D5 10 208779 79 1970 PDE4DH2 16 208780 80 1750 PDE4D1 18 208780 80 1664 PDE4D2 19 208780 80 2085 PDE4D3 20 208780 80 2841 PDE4D4 15 208780 80 2344 PDE4D5 10 208780 80 2142 PDE4DH2 16 208781 81 2909 PDE4D4 15 208781 81 2412 PDE4D5 10 208781 81 2210 PDE4DH2 16 208782 82 3000 PDE4D4 15 208782 82 2503 PDE4D5 10 208782 82 2301 PDE4DH2 16 208783 83 3087 PDE4D4 15 208783 83 2590 PDE4D5 10 208783 83 2388 PDE4DH2 16 208784 84 3168 PDE4D4 15 208784 84 2671 PDE4D5 10 208784 84 2469 PDE4DH2 16 208785 85 3301 PDE4D4 15 208785 85 2804 PDE4D5 10 208785 85 2602 PDE4DH2 16 208786 86 3525 PDE4D4 15 208786 86 3028 PDE4D5 10 208786 86 2826 PDE4DH2 16 208787 87 3804 PDE4D4 15 208787 87 3307 PDE4D5 10 208787 87 3105 PDE4DH2 16 208788 88 49 PDE4DH2 16 208789 89 56 PDE4D3 20 208789 89 113 PDE4DH2 16 208790 90 112 PDE4D3 20 208790 90 169 PDE4DH2 16 208793 93 463 PDE4DN2 11 208794 94 26 PDE4DH1 12 208795 95 195 PDE4DH1 12 208796 96 202 PDE4DH1 12

[0287]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 100 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 atgcattctg cccccaagga 20 <210> SEQ ID NO 3 <211> LENGTH: 5706 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (109)...(1923) <400> SEQUENCE: 3 ggaattccat tttccctccc ttttctccaa gcaaattttg tccacagtca acgacgggag 60 tccttcctgt atcgatccga cagcgattat gacctctctc caaagtct atg tcc cgg 117 Met Ser Arg 1 aac tcc tcc att gcc agt gat ata cac gga gat gac ttg att gtg act 165 Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr 5 10 15 cca ttt gct cag gtc ttg gcc agt ctg cga act gta cga aac aac ttt 213 Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe 20 25 30 35 gct gca tta act aat ttg caa gat cga gca cct agc aaa aga tca ccc 261 Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro 40 45 50 atg tgc aac caa cca tcc atc aac aaa gcc acc ata aca gag gag gcc 309 Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala 55 60 65 tac cag aaa ctg gcc agc gag acc ctg gag gag ctg gac tgg tgt ctg 357 Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu 70 75 80 gac cag cta gag acc cta cag acc agg cac tcc gtc agt gag atg gcc 405 Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala 85 90 95 tcc aac aag ttt aaa agg atg ctt aat cgg gag ctc acc cat ctc tct 453 Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser 100 105 110 115 gaa atg agt cgg tct gga aat caa gtg tca gag ttt ata tca aac aca 501 Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr 120 125 130 ttc tta gat aag caa cat gaa gtg gaa att cct tct cca act cag aag 549 Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys 135 140 145 gaa aag gag aaa aag aaa aga cca atg tct cag atc agt gga gtc aag 597 Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys 150 155 160 aaa ttg atg cac agc tct agt ctg act aat tca agt atc cca agg ttt 645 Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe 165 170 175 gga gtt aaa act gaa caa gaa gat gtc ctt gcc aag gaa cta gaa gat 693 Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp 180 185 190 195 gtg aac aaa tgg ggt ctt cat gtt ttc aga ata gca gag ttg tct ggt 741 Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly 200 205 210 aac cgg ccc ttg act gtt atc atg cac acc att ttt cag gaa cgg gat 789 Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp 215 220 225 tta tta aaa aca ttt aaa att cca gta gat act tta att aca tat ctt 837 Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu 230 235 240 atg act ctc gaa gac cat tac cat gct gat gtg gcc tat cac aac aat 885 Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn 245 250 255 atc cat gct gca gat gtt gtc cag tct act cat gtg cta tta tct aca 933 Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr 260 265 270 275 cct gct ttg gag gct gtg ttt aca gat ttg gag att ctt gca gca att 981 Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile 280 285 290 ttt gcc agt gca ata cat gat gta gat cat cct ggt gtg tcc aat caa 1029 Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln 295 300 305 ttt ctg atc aat aca aac tct gaa ctt gcc ttg atg tac aat gat tcc 1077 Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser 310 315 320 tca gtc tta gag aac cat cat ttg gct gtg ggc ttt aaa ttg ctt cag 1125 Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln 325 330 335 gaa gaa aac tgt gac att ttc cag aat ttg acc aaa aaa caa aga caa 1173 Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln 340 345 350 355 tct tta agg aaa atg gtc att gac atc gta ctt gca aca gat atg tca 1221 Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser 360 365 370 aaa cac atg aat cta ctg gct gat ttg aag act atg gtt gaa act aag 1269 Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys 375 380 385 aaa gtg aca agc tct gga gtt ctt ctt ctt gat aat tat tcc gat agg 1317 Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg 390 395 400 att cag gtt ctt cag aat atg gtg cac tgt gca gat ctg agc aac cca 1365 Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro 405 410 415 aca aag cct ctc cag ctg tac cgc cag tgg acg gac cgg ata atg gag 1413 Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu 420 425 430 435 gag ttc ttc ccc caa gga gac cga gag agg gaa cgt ggc atg gag ata 1461 Glu Phe Phe Pro Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile 440 445 450 agc ccc atg tgt gac aag cac aat gct tcc gtg gaa aaa tca cag gtg 1509 Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val 455 460 465 ggc ttc ata gac tat att gtt cat ccc ctc tgg gag aca tgg gca gac 1557 Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp 470 475 480 ctc gtc cac cct gac gcc cag gat att ttg gac act ttg gag gac aat 1605 Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn 485 490 495 cgt gaa tgg tac cag agc aca atc cct cag agc ccc tct cct gca cct 1653 Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro 500 505 510 515 gat gac cca gag gag ggc cgg cag ggt caa act gag aaa ttc cag ttt 1701 Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe 520 525 530 gaa cta act tta gag gaa gat ggt gag tca gac acg gaa aag gac agt 1749 Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser 535 540 545 ggc agt caa gtg gaa gaa gac act agc tgc agt gac tcc aag act ctt 1797 Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu 550 555 560 tgt act caa gac tca gag tct act gaa att ccc ctt gat gaa cag gtt 1845 Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val 565 570 575 gaa gag gag gca gta ggg gaa gaa gag gaa agc cag cct gaa gcc tgt 1893 Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys 580 585 590 595 gtc ata gat gat cgt tct cct gac acg taa cagtgcaaaa actttcatgc 1943 Val Ile Asp Asp Arg Ser Pro Asp Thr 600 cttttttttt tttaagtaga aaaattgttt ccaaagtgca tgtcacatgc cacaaccacg 2003 gtcacacctc actgtcatct gccaggacgt ttgttgaaca aaactgacct tgactactca 2063 gtccagcgct caggaatatc gtaaccagtt ttttcacctc catgtcatcc gagcaaggtg 2123 gacatcttca cgaacagcgt ttttaacaaa atttcagctt ggtagagctg acaaagcaga 2183 taaaatctac tccaaattat tttcaagaga gtgtgactca tcaggcagcc caaaagttta 2243 ttggacttgg ggtttctatt cctttttatt tgtttgcaat attttcagaa gaaaggcatt 2303 gcacagagtg aacttaatgg acgaagcaac aaatatgtca agaacaggac atagcacgaa 2363 tctgttacca gtaggaggag gatgagccac agaaattgca taattttcta atttcaagtc 2423 ttcctgatac atgactgaat agtgtggttc agtgagctgc actgacctct acattttgta 2483 tgatatgtaa aacagatttt ttgtagagct tacttttatt attaaatgta ttgaggtatt 2543 atatttaaaa aaaactatgt tcagaacttc atctgccact ggttattttt ttctaaggag 2603 taacttgcaa gttttcagta caaatctgtg ctacactgga taaaaatcta atttatgaat 2663 tttacttgca ccttatagtt catagcaatt aactgatttg tagtgattca ttgtttgttt 2723 tatataccaa tgacttccat attttaaaag agaaaaacaa ctttatgttg caggaaaccc 2783 tttttgtaag tctttattat tcactttgca ttttgtttca ctctttccag ataagcagag 2843 ttgctcttca ccagtgtttt tcttcatgtg caaagtgact atttgttcta taatactttt 2903 atgtgtgtta tatcaaatgt gtcttaagct tcatgcaaac tcagtcatca gttcgtgttg 2963 tctgaagcaa gtgggaaata tataaatacc cagtagctaa aatggtcagt cttttttaga 3023 tgttttccta cttagtatct cctaataacg ttttgctgtg tcactagatg ttcatttcac 3083 aagtgcatgt ctttctaata atccacacat ttcatgctct aataatccac acatttcatg 3143 ctcattttta ttgtttttac agccagttat agcaagaaaa aggtttttcc ccttgtgctg 3203 ctttataatt tagcgtgtgt ctgaacctta tccatgtttg ctagatgagg tcttgtcaaa 3263 tatatcacta ccattgtcac cggtgaaaag aaacaggtag ttaagttagg gttaacattc 3323 atttcaacca cgaggttgta tatcatgact agcttttact cttggtttac agagaaaagt 3383 taaacaacca actaggcagt ttttaagaat attaacaata tattaacaaa caccaataca 3443 actaatccta tttggtttta atgatttcac catgggatta agaactatat caggaacatc 3503 cctgagaaac ggctttaagt gtagcaacta ctcttcctta atggacagcc acataacgtg 3563 taggaagtcc tttatcactt atcctcgatc cataagcata tcttgcagag gggaactact 3623 tctttaaaca catggaggga aagaagatga tgccactggc accagagggt tagtactgtg 3683 atgcatccta aaatatttat tatattggta aaaattctgg ttaaataaaa aattagagat 3743 cactcttggc tgatttcagc accaggaact gtattacagt tttagagatt aattcctagt 3803 gtttacctga ttatagcagt tggcatcatg gggcatttaa ttctgacttt atccccacgt 3863 cagccttaat aaagtcttct ttaccttctc tatgaagact ttaaagccca aataatcatt 3923 tttcacattg atattcaaga attgagatag atagaagcca aagtgggtat ctgacaagtg 3983 gaaaatcaaa cgtttaagaa gaattacaac tctgaaaagc atttatatgt ggaacttctc 4043 aaggagcctc ctggggactg gaaagtaagt catcagccag gcaaatgact catgctgaag 4103 agagtcccca tttcagtccc ctgagatcta gctgatgctt agatcctttg aaataaaaat 4163 tatgtcttta taactctgat cttttacata aagcagaaga ggaatcaact agttaattgc 4223 aaggtttcta ctctgtttcc tctgtaaaga tcagatggta atctttcaaa taagaaaaaa 4283 ataaagacgt atgtttgacc aagtagtttc acaagaatat ttgggaactt gtttctttta 4343 attttatttg tccctgagtg aagtctagaa agaaaggtaa agagtctaga gtttattcct 4403 ctttccaaaa cattctcatt cctctcctcc ctacacttag tatttccccc acagagtgcc 4463 tagaatctta ataatgaata aaataaaaag cagcaatatg tcattaacaa atccagacct 4523 gaaagggtaa agggtttata actgcactaa taaagagagg ctcttttttt ttcttccagt 4583 ttgttggttt ttaatggtac cgtgttgtaa agatacccac taatggacaa tcaaattgca 4643 gaaaaggctc aatatccaag agacagggac taatgcactg tacaatctgc ttatccttgc 4703 ccttctctct tgccaaagtg tgcttcagaa atatatactg ctttaaaaaa gaataaaaga 4763 atatcctttt acaagtggct ttacatttcc taaaatgcca taagaaaatg caatatctgg 4823 gtactgtatg gggaaaaaaa tgtccaagtt tgtgtaaaac cagtgcattt cagcttgcaa 4883 gttactgaac acaataatgc tgttttaatt ttgttttata tcagttaaaa ttcacaataa 4943 tgtagataga acaaattaca gacaaggaaa gaaaaaactt gaatgaaatg gattttacag 5003 aaagctttat gataattttt gaatgcatta tttatttttt gtgccatgca ttttttttct 5063 caccaaatga ccttacctgt aatacagtct tgtttgtctg tttacaacca tgtatttatt 5123 gcaatgtaca tactgtaatg ttaattgtaa attatctgtt cttattaaaa catcatccca 5183 tgatggggtg gtgttgatat atttggaaac tcttggtgag agaatgaatg gtgtgtatac 5243 atactctgta catttttctt ttctcctgta atatagtctt gtcaccttag agcttgttta 5303 tggaagattc aagaaaacta taaaatactt aaagatatat aaatttaaaa aaacatagct 5363 gcaggtcttt ggtcccaggg ctgtgcctta actttaacca atattttctt ctgttttgct 5423 gcatttgaaa ggtaacagtg gagctagggc tgggcatttt acatccaggc ttttaattga 5483 ttagaattct gccaataggt ggattttaca aaaccacaga caacctctga aagattctga 5543 gacccttttg agacagaagc tcttaagtac ttcttgccag ggagcagcac tgcatgtgtg 5603 atggttgttt gccatctgtt gatcaggaac tacttcagct acttgcattt gattatttcc 5663 tttttttttt tttttaactc ggaaacacaa ctgggggaat tcc 5706 <210> SEQ ID NO 4 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 4 agcgctcagg aatatcgtaa cc 22 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 aaaacgctgt tcgtgaagat gtc 23 <210> SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 6 tcacctccat gtcatccgag ca 22 <210> SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 7 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaagatggtg atgggatttc 20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 9 caagcttccc gttctcagcc 20 <210> SEQ ID NO 10 <211> LENGTH: 3332 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (118)...(2355) <400> SEQUENCE: 10 gaattcggca cgagcagcag caggctcaga cctgcttccc tggacatttc cgggaccgtg 60 agcgagggaa ccacgttgcc ctggattctt gccagctgta caaagttgac caggaaa 117 atg gct cag cag aca agc ccg gac act tta aca gta cct gaa gtg gat 165 Met Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu Val Asp 1 5 10 15 aat ccg cat tgt cca aac ccg tgg ctg aac gaa gac ctt gtg aaa tcc 213 Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val Lys Ser 20 25 30 ttg cga gaa aac ctg ttg cag cat gag aag tcc aag aca gcg agg aaa 261 Leu Arg Glu Asn Leu Leu Gln His Glu Lys Ser Lys Thr Ala Arg Lys 35 40 45 tcg gtt tct ccc aag ctc tct cca gtg atc tct ccg aga aat tcc ccc 309 Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn Ser Pro 50 55 60 agg ctt ctg cgc aga atg ctt ctc agc agc aac atc ccc aaa cag cgg 357 Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys Gln Arg 65 70 75 80 cgt ttc acg gtg gca cat aca tgt ttt gat gtg gac aat ggc aca tct 405 Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly Thr Ser 85 90 95 gcg gga cgg agt ccc ttg gat ccc atg acc agc cca gga tcc ggg cta 453 Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu 100 105 110 att ctc caa gca aat ttt gtc cac agt caa cga cgg gag tcc ttc ctg 501 Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu 115 120 125 tat cga tcc gac agc gat tat gac ctc tct cca aag tct atg tcc cgg 549 Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg 130 135 140 aac tcc tcc att gcc agt gat ata cac gga gat gac ttg att gtg act 597 Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr 145 150 155 160 cca ttt gct cag gtc ttg gcc agt ctg cga act gta cga aac aac ttt 645 Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe 165 170 175 gct gca tta act aat ttg caa gat cga gca cct agc aaa aga tca ccc 693 Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro 180 185 190 atg tgc aac caa cca tcc atc aac aaa gcc acc ata aca gag gag gcc 741 Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala 195 200 205 tac cag aaa ctg gcc agc gag acc ctg gag gag ctg gac tgg tgt ctg 789 Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu 210 215 220 gac cag cta gag acc cta cag acc agg cac tcc gtc agt gag atg gcc 837 Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala 225 230 235 240 tcc aac aag ttt aaa agg atg ctt aat cgg gag ctc acc cat ctc tct 885 Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser 245 250 255 gaa atg agt cgg tct gga aat caa gtg tca gag ttt ata tca aac aca 933 Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr 260 265 270 ttc tta gat aag caa cat gaa gtg gaa att cct tct cca act cag aag 981 Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys 275 280 285 gaa aag gag aaa aag aaa aga cca atg tct cag atc agt gga gtc aag 1029 Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys 290 295 300 aaa ttg atg cac agc tct agt ctg act aat tca agt atc cca agg ttt 1077 Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe 305 310 315 320 gga gtt aaa act gaa caa gaa gat gtc ctt gcc aag gaa cta gaa gat 1125 Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp 325 330 335 gtg aac aaa tgg ggt ctt cat gtt ttc aga ata gca gag ttg tct ggt 1173 Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly 340 345 350 aac cgg ccc ttg act gtt atc atg cac acc att ttt cag gaa cgg gat 1221 Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp 355 360 365 tta tta aaa aca ttt aaa att cca gta gat act tta att aca tat ctt 1269 Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu 370 375 380 atg act ctc gaa gac cat tac cat gct gat gtg gcc tat cac aac aat 1317 Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn 385 390 395 400 atc cat gct gca gat gtt gtc cag tct act cat gtg cta tta tct aca 1365 Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr 405 410 415 cct gct ttg gag gct gtg ttt aca gat ttg gag att ctt gca gca att 1413 Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile 420 425 430 ttt gcc agt gca ata cat gat gta gat cat cct ggt gtg tcc aat caa 1461 Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln 435 440 445 ttt ctg atc aat aca aac tct gaa ctt gcc ttg atg tac aat gat tcc 1509 Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser 450 455 460 tca gtc tta gag aac cat cat ttg gct gtg ggc ttt aaa ttg ctt cag 1557 Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln 465 470 475 480 gaa gaa aac tgt gac att ttc cag aat ttg acc aaa aaa caa aga caa 1605 Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln 485 490 495 tct tta agg aaa atg gtc att gac atc gta ctt gca aca gat atg tca 1653 Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser 500 505 510 aaa cac atg aat cta ctg gct gat ttg aag act atg gtt gaa act aag 1701 Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys 515 520 525 aaa gtg aca agc tct gga gtt ctt ctt ctt gat aat tat tcc gat agg 1749 Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg 530 535 540 att cag gtt ctt cag aat atg gtg cac tgt gca gat ctg agc aac cca 1797 Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro 545 550 555 560 aca aag cct ctc cag ctg tac cgc cag tgg acg gac cgg ata atg gag 1845 Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu 565 570 575 gag ttc ttc cgc caa gga gac cga gag agg gaa cgt ggc atg gag ata 1893 Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile 580 585 590 agc ccc atg tgt gac aag cac aat gct tcc gtg gaa aaa tca cag gtg 1941 Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val 595 600 605 ggc ttc ata gac tat att gtt cat ccc ctc tgg gag aca tgg gca gac 1989 Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp 610 615 620 ctc gtc cac cct gac gcc cag gat att ttg gac act ttg gag gac aat 2037 Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn 625 630 635 640 cgt gaa tgg tac cag agc aca atc cct cag agc ccc tct cct gca cct 2085 Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro 645 650 655 gat gac cca gag gag ggc cgg cag ggt caa act gag aaa ttc cag ttt 2133 Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe 660 665 670 gaa cta act tta gag gaa gat ggt gag tca gac acg gaa aag gac agt 2181 Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser 675 680 685 ggc agt caa gtg gaa gaa gac act agc tgc agt gac tcc aag act ctt 2229 Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu 690 695 700 tgt act caa gac tca gag tct act gaa att ccc ctt gat gaa cag gtt 2277 Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val 705 710 715 720 gaa gag gag gca gta ggg gaa gaa gag gaa agc cag cct gaa gcc tgt 2325 Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys 725 730 735 gtc ata gat gat cgt tct cct gac acg taa cagtgcaaaa actttcatgc 2375 Val Ile Asp Asp Arg Ser Pro Asp Thr 740 745 cttttttttt tttaagtaga aaaattgttt ccaaagtgca tgtcacatgc cacaaccacg 2435 gtcacacctc actgtcatct gccaggacgt ttgttgaaca aaactgacct tgactactca 2495 gtccagcgct caggaatatc gtaaccagtt ttttcacctc catgttcatc cgagcaaggt 2555 ggacatcttc acgaacagcg tttttaacaa gatttcagct tggtagagct gacaaagcag 2615 ataaaatcta ctccaaatta ttttcaagag agtgtgactc atcaggcagc ccaaaagttt 2675 attggacttg gggtttctat tcctttttat ttgtttgcaa tattttcaga agaaaggcat 2735 tgcacagagt gaacttaatg gacgaagcaa caaatatgtc aagaacagga catagcacga 2795 atctgttacc agtaggagga ggatgagcca cagaaattgc ataattttct aatttcaagt 2855 cttcctgata catgactgaa tagtgtggtt cagtgagctg cactgacctc tacattttgt 2915 atgatatgta aaacagattt tttgtagagc ttacttttat tattaaatgt attgaggtat 2975 tatatttaaa aaaaactatg ttcagaactt catctgccac tggttatttt tttctaagga 3035 gtaacttgca agttttcagt acaaatctgt gctacactgg ataaaaatct aatttatgaa 3095 ttttacttgc accttatagt tcatagcaat taactgattt gtagtgattc attgtttgtt 3155 ttatatacca atgacttcca tattttaaaa gagaaaaaca actttatgtt gcaggaaacc 3215 ctttttgtaa gtctttatta tttactttgc attttgtttc actctttcca gataagcaga 3275 gttgctcttc accagtgttt ttcttcatgt gcaaagtgac tatttgttct ataatac 3332 <210> SEQ ID NO 11 <211> LENGTH: 512 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (24)...(476) <400> SEQUENCE: 11 agcgctacct gtactgtcgc gcc atg gac cgc acc tcc tac gcg gtg gag acc 53 Met Asp Arg Thr Ser Tyr Ala Val Glu Thr 1 5 10 ggc cac cgg ccc ggc ctg aag aaa tcc agg atg tcc tgg ccc tcc tcg 101 Gly His Arg Pro Gly Leu Lys Lys Ser Arg Met Ser Trp Pro Ser Ser 15 20 25 ttc cag gga ctc agg cgt ttt gat gtg gac aat ggc aca tct gcg gga 149 Phe Gln Gly Leu Arg Arg Phe Asp Val Asp Asn Gly Thr Ser Ala Gly 30 35 40 cgg agt ccc ttg gat ccc atg acc agc cca gga tcc ggg cta att ctc 197 Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu Ile Leu 45 50 55 caa gca aat ttt gtc cac agt caa cga cgg gag tcc ttc ctg tat cga 245 Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg 60 65 70 tcc gac agc gat tat gac ctc tct cca aag tct atg tcc cgg aac tcc 293 Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg Asn Ser 75 80 85 90 tcc att gcc agt gat ata cac gga gat gac ttg att gtg act cca ttt 341 Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr Pro Phe 95 100 105 gct cag gtc ttg gcc agt ctg cga act gta cga aac aac ttt gct gca 389 Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe Ala Ala 110 115 120 tta act aat ttg caa gat cga gca cct agc aaa aga tca ccc atg tgc 437 Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro Met Cys 125 130 135 aac caa cca tcc atc aac aaa gcc acc ata aca gtt taa aaggatgctt 486 Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Val 140 145 150 aatcgggagc tcacccatct ctctga 512 <210> SEQ ID NO 12 <211> LENGTH: 659 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 150 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 566 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 569 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 593 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 625 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 631 <223> OTHER INFORMATION: unknown <400> SEQUENCE: 12 aaaggaaatg ccctcactca gctacaagtg cctcctgctc cgtgcggggc ctcagggccc 60 cagagccccg caccagctct gacttctcgt ggttcctggg gatcttggca tcgtccttaa 120 aaatggcttt tgtttgggat cctctgggan ccacggtgcc aggaccatct acaagagcca 180 aatcaagatt gcgtttctca aagtcctaca gttttgatgt ggacaatggc acatctgcgg 240 gacggagtcc cttggatccc atgaccagcc caggatccgg gctaattctc caagcaaatt 300 ttgtccacag tcaacgacgg gagtccttcc tgtatcgatc cgacagcgat tatgacctct 360 ctccaaagtc tatgtcccgg aactcctcca ttgccagtga tatacacgga gatgacttga 420 ttgtgactcc atttgctcag gtcttggcca gtctgcgaac tgtacgaaac aactttgctg 480 cattaactaa tttgcaagat cgagcaccta gcaaaagatc acccatgtgc aaccaaccat 540 ccatcaacaa agccaccata acagangang cctaccagaa actggccagc ganacctgga 600 agaactggac tggtgtctgg accanctaga nacctacaga ccaggcactc cgtcagtga 659 <210> SEQ ID NO 13 <211> LENGTH: 1040 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (128)...(787) <400> SEQUENCE: 13 gaggcaggcg actgaatgca ctaacagcag caggctcaga cctgcttccc tggacatttc 60 cgggaccgtg agcgagggaa ccacgttgcc ctggattctt gccagctgta caaagttgac 120 caggaaa atg gct cag cag aca agc ccg gac act tta aca gta cct gaa 169 Met Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu 1 5 10 gtg gat aat ccg cat tgt cca aac ccg tgg ctg aac gaa gac ctt gtg 217 Val Asp Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val 15 20 25 30 aaa tcc ttg cga gaa aac ctg ttg cag cat gag aag tcc aag aca gcg 265 Lys Ser Leu Arg Glu Asn Leu Leu Gln His Glu Lys Ser Lys Thr Ala 35 40 45 agg aaa tcg gtt tct ccc aag ctc tct cca gtg atc tct ccg aga aat 313 Arg Lys Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn 50 55 60 tcc ccc agg ctt ctg cgc aga atg ctt ctc agc agc aac atc ccc aaa 361 Ser Pro Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys 65 70 75 cag cgg cgt ttc acg gtg gca cat aca tgt ttt gat gtg gac aat ggc 409 Gln Arg Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly 80 85 90 aca tct gcg gga cgg agt ccc ttg gat ccc atg acc agc cca gga tcc 457 Thr Ser Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser 95 100 105 110 ggg cta att ctc caa gca aat ttt gtc cac agt caa cga cgg gag tcc 505 Gly Leu Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser 115 120 125 ttc ctg tat cga tcc gac agc gat tat gac ctc tct cca aag tct atg 553 Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met 130 135 140 tcc cgg aac tcc tcc att gcc agt gat ata cac gga gat gac ttg att 601 Ser Arg Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile 145 150 155 gtg act cca ttt gct cag gtc ttg gcc agt ctg cga act gta cga aac 649 Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn 160 165 170 aac ttt gct gca tta act aat ttg caa gat cga gca cct agc aaa aga 697 Asn Phe Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg 175 180 185 190 tca ccc atg tgc aac caa cca tcc atc aac aaa gcc acc ata aca gga 745 Ser Pro Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Gly 195 200 205 tcc tgg atg gaa ttg aac cca tat acg tta ctt gac atg tga aacacgtgtg 797 Ser Trp Met Glu Leu Asn Pro Tyr Thr Leu Leu Asp Met 210 215 accctggcag atgatttggc tgaccttgaa aactacagct gtttagtcac tttgaaaaca 857 atgcaataca agtgatttac taggcttcag ttttaaccat tttatgctga ctggtggaaa 917 ttctaaccct tcagaaagaa aaaaaaattg tgaagcaatg gaaatgtacc atcctgggta 977 ttaatgtcat taattttcaa tacattttct cgcaaaaaaa aaaaaaaaaa aaaaaaaaaa 1037 aaa 1040 <210> SEQ ID NO 14 <211> LENGTH: 772 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 205 <223> OTHER INFORMATION: unknown <400> SEQUENCE: 14 gtttgctgca tttgaaaggt aacagtggag ctagggctgg gcattttaca tccaggcttt 60 taattgatta gaattctgcc aataggtgga ttttacaaaa ccacagacaa cctctgaaag 120 attctgagac cctttgagac agaagctctt aagtacttct tgccaggagc agcactgcat 180 gtgtgatggt tgtttgccat ctgtngatca ggaactactt cagctacttg catttgatta 240 tttccttttt tttttttttt aactcggaaa cacaacgggg gaaatatatt ctttcccagt 300 gattataaac aatctttttc tttttttata agtcctttgg gcttctagag ctcataggaa 360 aagtggactt gatttgaaat gggagccaga gtttactcgt ggtgggttat ctattcatca 420 gcttcctgac atgttaagag aatacattaa agagaaaata ctggtttttt aatcctaaaa 480 tttttcttcc actaagataa accaaatgtg cttacatatt tgtaaaccca tctatttaaa 540 cgcaaaggtg ggttgatgtc agtttacata gcagaaagca ttcactatcc tctaagattg 600 gtgttctgga aaactttcat ggtttagaat tttaaaattt cacctggtac aaatggcagg 660 ccctaaagca ggaaacattt ataaggggtt attgtgggaa aatccttcca gtatttggcc 720 agccctgaat atgtggcttc agtgaagaag atctttttta gaaatggcta ta 772 <210> SEQ ID NO 15 <211> LENGTH: 3829 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 46 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 47 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (423)...(2852) <400> SEQUENCE: 15 gaattccctg gatacagcct ttttatgact tttacttcct ttatanncaa attccaacgt 60 cttctcattc ctccaccagg sctgtgccaa cctgggccca acccaaggsc ctcactaaac 120 catccaatca gtaggagcca tagactactt tatttagcca aagcaaaaat gagtcaactg 180 aattctgttt ttccatttac ttctgtctgt ttttccttcc tcttgccacc ctcagtgcca 240 caagagggga cccctctcgg tagccctgag gctctggcgc cttcaagtga gaagctaagc 300 accagcctct gctgggctgc agaagcggcg gcggcggcag cagcagcagc agcatcagga 360 aggctctcgg gccagcgcgg tgaacccggg ctgggcagca ggtcgcggag ccgcgagcca 420 gg atg gag gca gag ggc agc agc gcg ccg gcc cgg gcg ggc agc gga 467 Met Glu Ala Glu Gly Ser Ser Ala Pro Ala Arg Ala Gly Ser Gly 1 5 10 15 gag ggc agc gac agc gcc ggc ggg gcc acg ctc aaa gcc ccc aag cat 515 Glu Gly Ser Asp Ser Ala Gly Gly Ala Thr Leu Lys Ala Pro Lys His 20 25 30 ctc tgg agg cac gag cag cac cac cag tac ccg ctc cgg cag ccc cag 563 Leu Trp Arg His Glu Gln His His Gln Tyr Pro Leu Arg Gln Pro Gln 35 40 45 ttc cgc ctc ctg cat ccc cat cac cac ctg ccc ccg ccg ccg cca ccc 611 Phe Arg Leu Leu His Pro His His His Leu Pro Pro Pro Pro Pro Pro 50 55 60 tcg ccc cag ccc cag ccc cag tgt ccg cta cag ccg ccg ccg ccg ccc 659 Ser Pro Gln Pro Gln Pro Gln Cys Pro Leu Gln Pro Pro Pro Pro Pro 65 70 75 ccc ctg ccg ccg ccc ccg ccg ccg ccc ggg gct gcc cgc ggc cgc tac 707 Pro Leu Pro Pro Pro Pro Pro Pro Pro Gly Ala Ala Arg Gly Arg Tyr 80 85 90 95 gcc tcg agc ggg gcc acc ggc cgc gtc cgg cat cgc ggc tac tcg gac 755 Ala Ser Ser Gly Ala Thr Gly Arg Val Arg His Arg Gly Tyr Ser Asp 100 105 110 acc gag cgc tac ctg tac tgt cgc gcc atg gac cgc acc tcc tac gcg 803 Thr Glu Arg Tyr Leu Tyr Cys Arg Ala Met Asp Arg Thr Ser Tyr Ala 115 120 125 gtg gag acc ggc cac cgg ccc ggc ctg aag aaa tcc agg atg tcc tgg 851 Val Glu Thr Gly His Arg Pro Gly Leu Lys Lys Ser Arg Met Ser Trp 130 135 140 ccc tcc tcg ttc cag gga ctc agg cgt ttt gat gtg gac aat ggc aca 899 Pro Ser Ser Phe Gln Gly Leu Arg Arg Phe Asp Val Asp Asn Gly Thr 145 150 155 tct gcg gga cgg agt ccc ttg gat ccc atg acc agc cca gga tcc ggg 947 Ser Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly 160 165 170 175 cta att ctc caa gca aat ttt gtc cac agt caa cga cgg gag tcc ttc 995 Leu Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe 180 185 190 ctg tat cga tcc gac agc gat tat gac ctc tct cca aag tct atg tcc 1043 Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser 195 200 205 cgg aac tcc tcc att gcc agt gat ata cac gga gat gac ttg att gtg 1091 Arg Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val 210 215 220 act cca ttt gct cag gtc ttg gcc agt ctg cga act gta cga aac aac 1139 Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn 225 230 235 ttt gct gca tta act aat ttg caa gat cga gca cct agc aaa aga tca 1187 Phe Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser 240 245 250 255 ccc atg tgc aac caa cca tcc atc aac aaa gcc acc ata aca gag gag 1235 Pro Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu 260 265 270 gcc tac cag aaa ctg gcc agc gag acc ctg gag gag ctg gac tgg tgt 1283 Ala Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys 275 280 285 ctg gac cag cta gag acc cta cag acc agg cac tcc gtc agt gag atg 1331 Leu Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met 290 295 300 gcc tcc aac aag ttt aaa agg atg ctt aat cgg gag ctc acc cat ctc 1379 Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu 305 310 315 tct gaa atg agt cgg tct gga aat caa gtg tca gag ttt ata tca aac 1427 Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn 320 325 330 335 aca ttc tta gat aag caa cat gaa gtg gaa att cct tct cca act cag 1475 Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln 340 345 350 aag gaa aag gag aaa aag aaa aga cca atg tct cag atc agt gga gtc 1523 Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val 355 360 365 aag aaa ttg atg cac agc tct agt ctg act aat tca agt atc cca agg 1571 Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg 370 375 380 ttt gga gtt aaa act gaa caa gaa gat gtc ctt gcc aag gaa cta gaa 1619 Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu 385 390 395 gat gtg aac aaa tgg ggt ctt cat gtt ttc aga ata gca gag ttg tct 1667 Asp Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser 400 405 410 415 ggt aac cgg ccc ttg act gtt atc atg cac acc att ttt cag gaa cgg 1715 Gly Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg 420 425 430 gat tta tta aaa aca ttt aaa att cca gta gat act tta att aca tat 1763 Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr 435 440 445 ctt atg act ctc gaa gac cat tac cat gct gat gtg gcc tat cac aac 1811 Leu Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn 450 455 460 aat atc cat gct gca gat gtt gtc cag tct act cat gtg cta tta tct 1859 Asn Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser 465 470 475 aca cct gct ttg gag gct gtg ttt aca gat ttg gag att ctt gca gca 1907 Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala 480 485 490 495 att ttt gcc agt gca ata cat gat gta gat cat cct ggt gtg tcc aat 1955 Ile Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn 500 505 510 caa ttt ctg atc aat aca aac tct gaa ctt gcc ttg atg tac aat gat 2003 Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp 515 520 525 tcc tca gtc tta gag aac cat cat ttg gct gtg ggc ttt aaa ttg ctt 2051 Ser Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu 530 535 540 cag gaa gaa aac tgt gac att ttc cag aat ttg acc aaa aaa caa aga 2099 Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg 545 550 555 caa tct tta agg aaa atg gtc att gac atc gta ctt gca aca gat atg 2147 Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met 560 565 570 575 tca aaa cac atg aat cta ctg gct gat ttg aag act atg gtt gaa act 2195 Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr 580 585 590 aag aaa gtg aca agc tct gga gtt ctt ctt ctt gat aat tat tcc gat 2243 Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp 595 600 605 agg att cag gtt ctt cag aat atg gtg cac tgt gca gat ctg agc aac 2291 Arg Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn 610 615 620 cca aca aag cct ctc cag ctg tac cgc cag tgg acg gac cgg ata atg 2339 Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met 625 630 635 gag gag ttc ttc cgc caa gga gac cga gag agg gaa cgt ggc atg gag 2387 Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu 640 645 650 655 ata agc ccc atg tgt gac aag cac aat gct tcc gtg gaa aaa tca cag 2435 Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln 660 665 670 gtg ggc ttc ata gac tat att gtt cat ccc ctc tgg gag aca tgg gca 2483 Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala 675 680 685 gac ctc gtc cac cct gac gcc cag gat att ttg gac act ttg gag gac 2531 Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp 690 695 700 aat cgt gaa tgg tac cag agc aca atc cct cag agc ccc tct cct gca 2579 Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala 705 710 715 cct gat gac cca gag gag ggc cgg cag ggt caa act gag aaa ttc cag 2627 Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln 720 725 730 735 ttt gaa cta act tta gag gaa gat ggt gag tca gac acg gaa aag gac 2675 Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp 740 745 750 agt ggc agt caa gtg gaa gaa gac act agc tgc agt gac tcc aag act 2723 Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr 755 760 765 ctt tgt act caa gac tca gag tct act gaa att ccc ctt gat gaa cag 2771 Leu Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln 770 775 780 gtt gaa gag gag gca gta ggg gaa gaa gag gaa agc cag cct gaa gcc 2819 Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala 785 790 795 tgt gtc ata gat gat cgt tct cct gac acg taa cagtgcaaaa actttcatgc 2872 Cys Val Ile Asp Asp Arg Ser Pro Asp Thr 800 805 cttttttttt tttaagtaga aaaattgttt ccaaagtgca tgtcacatgc cacaaccacg 2932 gtcacacctc actgtcatct gccaggacgt ttgttgaaca aaactgacct tgactactca 2992 gtccagcgct caggaatatc gtaaccagtt ttttcacctc catgttcatc cgagcaaggt 3052 ggacatcttc acgaacagcg tttttaacaa gatttcagct tggtagagct gacaaagcag 3112 ataaaatcta ctccaaatta ttttcaagag agtgtgactc atcaggcagc ccaaaagttt 3172 attggacttg gggtttctat tcctttttat ttgtttgcaa tattttcaga agaaaggcat 3232 tgcacagagt gaacttaatg gacgaagcaa caaatatgtc aagaacagga catagcacga 3292 atctgttacc agtaggagga ggatgagcca cagaaattgc ataattttct aatttcaagt 3352 cttcctgata catgactgaa tagtgtggtt cagtgagctg cactgacctc tacattttgt 3412 atgatatgta aaacagattt tttgtagagc ttacttttat tattaaatgt attgaggtat 3472 tatatttaaa aaaaactatg ttcagaactt catctgccac tggttatttt tttctaagga 3532 gtaacttgca agttttcagt acaaatctgt gctacactgg ataaaaatct aatttatgaa 3592 ttttacttgc accttatagt tcatagcaat taactgattt gtagtgattc attgtttgtt 3652 ttatatacca atgacttcca tattttaaaa gagaaaaaca actttatgtt gcaggaaacc 3712 ctttttgtaa gtctttatta tttactttgc attttgtttc actctttcca gataagcaga 3772 gttgctcttc accagtgttt ttcttcatgt gcaaagtgac tatttgttct ataatac 3829 <210> SEQ ID NO 16 <211> LENGTH: 3130 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (132)...(2153) <400> SEQUENCE: 16 gaattcccaa tacttgttgc aataattgcc cacgatagct gctcaaacaa gagagttgga 60 attcatctgt aaaaatcact acatgtaacg taggagacaa gaaaaatatt aatgacagaa 120 gatctgcgaa c atg atg cac gtg aat aat ttt ccc ttt aga agg cat tcc 170 Met Met His Val Asn Asn Phe Pro Phe Arg Arg His Ser 1 5 10 tgg ata tgt ttt gat gtg gac aat ggc aca tct gcg gga cgg agt ccc 218 Trp Ile Cys Phe Asp Val Asp Asn Gly Thr Ser Ala Gly Arg Ser Pro 15 20 25 ttg gat ccc atg acc agc cca gga tcc ggg cta att ctc caa gca aat 266 Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu Ile Leu Gln Ala Asn 30 35 40 45 ttt gtc cac agt caa cga cgg gag tcc ttc ctg tat cga tcc gac agc 314 Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser 50 55 60 gat tat gac ctc tct cca aag tct atg tcc cgg aac tcc tcc att gcc 362 Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg Asn Ser Ser Ile Ala 65 70 75 agt gat ata cac gga gat gac ttg att gtg act cca ttt gct cag gtc 410 Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr Pro Phe Ala Gln Val 80 85 90 ttg gcc agt ctg cga act gta cga aac aac ttt gct gca tta act aat 458 Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe Ala Ala Leu Thr Asn 95 100 105 ttg caa gat cga gca cct agc aaa aga tca ccc atg tgc aac caa cca 506 Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro Met Cys Asn Gln Pro 110 115 120 125 tcc atc aac aaa gcc acc ata aca gag gag gcc tac cag aaa ctg gcc 554 Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala Tyr Gln Lys Leu Ala 130 135 140 agc gag acc ctg gag gag ctg gac tgg tgt ctg gac cag cta gag acc 602 Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr 145 150 155 cta cag acc agg cac tcc gtc agt gag atg gcc tcc aac aag ttt aaa 650 Leu Gln Thr Arg His Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys 160 165 170 agg atg ctt aat cgg gag ctc acc cat ctc tct gaa atg agt cgg tct 698 Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser 175 180 185 gga aat caa gtg tca gag ttt ata tca aac aca ttc tta gat aag caa 746 Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe Leu Asp Lys Gln 190 195 200 205 cat gaa gtg gaa att cct tct cca act cag aag gaa aag gag aaa aag 794 His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys Glu Lys Lys 210 215 220 aaa aga cca atg tct cag atc agt gga gtc aag aaa ttg atg cac agc 842 Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys Leu Met His Ser 225 230 235 tct agt ctg act aat tca agt atc cca agg ttt gga gtt aaa act gaa 890 Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly Val Lys Thr Glu 240 245 250 caa gaa gat gtc ctt gcc aag gaa cta gaa gat gtg aac aaa tgg ggt 938 Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val Asn Lys Trp Gly 255 260 265 ctt cat gtt ttc aga ata gca gag ttg tct ggt aac cgg ccc ttg act 986 Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg Pro Leu Thr 270 275 280 285 gtt atc atg cac acc att ttt cag gaa cgg gat tta tta aaa aca ttt 1034 Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe 290 295 300 aaa att cca gta gat act tta att aca tat ctt atg act ctc gaa gac 1082 Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met Thr Leu Glu Asp 305 310 315 cat tac cat gct gat gtg gcc tat cac aac aat atc cat gct gca gat 1130 His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile His Ala Ala Asp 320 325 330 gtt gtc cag tct act cat gtg cta tta tct aca cct gct ttg gag gct 1178 Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Glu Ala 335 340 345 gtg ttt aca gat ttg gag att ctt gca gca att ttt gcc agt gca ata 1226 Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ser Ala Ile 350 355 360 365 cat gat gta gat cat cct ggt gtg tcc aat caa ttt ctg atc aat aca 1274 His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr 370 375 380 aac tct gaa ctt gcc ttg atg tac aat gat tcc tca gtc tta gag aac 1322 Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser Val Leu Glu Asn 385 390 395 cat cat ttg gct gtg ggc ttt aaa ttg ctt cag gaa gaa aac tgt gac 1370 His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu Asn Cys Asp 400 405 410 att ttc cag aat ttg acc aaa aaa caa aga caa tct tta agg aaa atg 1418 Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu Arg Lys Met 415 420 425 gtc att gac atc gta ctt gca aca gat atg tca aaa cac atg aat cta 1466 Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys His Met Asn Leu 430 435 440 445 ctg gct gat ttg aag act atg gtt gaa act aag aaa gtg aca agc tct 1514 Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser 450 455 460 gga gtt ctt ctt ctt gat aat tat tcc gat agg att cag gtt ctt cag 1562 Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Gln 465 470 475 aat atg gtg cac tgt gca gat ctg agc aac cca aca aag cct ctc cag 1610 Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Gln 480 485 490 ctg tac cgc cag tgg acg gac cgg ata atg gag gag ttc ttc cgc caa 1658 Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Arg Gln 495 500 505 gga gac cga gag agg gaa cgt ggc atg gag ata agc ccc atg tgt gac 1706 Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp 510 515 520 525 aag cac aat gct tcc gtg gaa aaa tca cag gtg ggc ttc ata gac tat 1754 Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr 530 535 540 att gtt cat ccc ctc tgg gag aca tgg gca gac ctc gtc cac cct gac 1802 Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp 545 550 555 gcc cag gat att ttg gac act ttg gag gac aat cgt gaa tgg tac cag 1850 Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr Gln 560 565 570 agc aca atc cct cag agc ccc tct cct gca cct gat gac cca gag gag 1898 Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp Pro Glu Glu 575 580 585 ggc cgg cag ggt caa act gag aaa ttc cag ttt gaa cta act tta gag 1946 Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu Thr Leu Glu 590 595 600 605 gaa gat ggt gag tca gac acg gaa aag gac agt ggc agt caa gtg gaa 1994 Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser Gln Val Glu 610 615 620 gaa gac act agc tgc agt gac tcc aag act ctt tgt act caa gac tca 2042 Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Cys Thr Gln Asp Ser 625 630 635 gag tct act gaa att ccc ctt gat gaa cag gtt gaa gag gag gca gta 2090 Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu Glu Glu Ala Val 640 645 650 ggg gaa gaa gag gaa agc cag cct gaa gcc tgt gtc ata gat gat cgt 2138 Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val Ile Asp Asp Arg 655 660 665 tct cct gac acg taa cagtgcaaaa actttcatgc cttttttttt tttaagtaga 2193 Ser Pro Asp Thr * 670 aaaattgttt ccaaagtgca tgtcacatgc cacaaccacg gtcacacctc actgtcatct 2253 gccaggacgt ttgttgaaca aaactgacct tgactactca gtccagcgct caggaatatc 2313 gtaaccagtt ttttcacctc catgttcatc cgagcaaggt ggacatcttc acgaacagcg 2373 tttttaacaa gatttcagct tggtagagct gacaaagcag ataaaatcta ctccaaatta 2433 ttttcaagag agtgtgactc atcaggcagc ccaaaagttt attggacttg gggtttctat 2493 tcctttttat ttgtttgcaa tattttcaga agaaaggcat tgcacagagt gaacttaatg 2553 gacgaagcaa caaatatgtc aagaacagga catagcacga atctgttacc agtaggagga 2613 ggatgagcca cagaaattgc ataattttct aatttcaagt cttcctgata catgactgaa 2673 tagtgtggtt cagtgagctg cactgacctc tacattttgt atgatatgta aaacagattt 2733 tttgtagagc ttacttttat tattaaatgt attgaggtat tatatttaaa aaaaactatg 2793 ttcagaactt catctgccac tggttatttt tttctaagga gtaacttgca agttttcagt 2853 acaaatctgt gctacactgg ataaaaatct aatttatgaa ttttacttgc accttatagt 2913 tcatagcaat taactgattt gtagtgattc attgtttgtt ttatatacca atgacttcca 2973 tattttaaaa gagaaaaaca actttatgtt gcaggaaacc ctttttgtaa gtctttatta 3033 tttactttgc attttgtttc actctttcca gataagcaga gttgctcttc accagtgttt 3093 ttcttcatgt gcaaagtgac tatttgttct ataatac 3130 <210> SEQ ID NO 17 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 17 000 <210> SEQ ID NO 18 <211> LENGTH: 1770 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (4)...(1761) <400> SEQUENCE: 18 aat atg aag gag cag ccc tca tgt gcc ggc acc ggg cat ccg agc atg 48 Met Lys Glu Gln Pro Ser Cys Ala Gly Thr Gly His Pro Ser Met 1 5 10 15 gcg ggg tat ggc agg atg gcc ccc ttt gaa ctc gct agc gga ccc gtg 96 Ala Gly Tyr Gly Arg Met Ala Pro Phe Glu Leu Ala Ser Gly Pro Val 20 25 30 aag cgc ttg aga act gag tcc ccc ttt ccc tgt ctc ttc gca gag gag 144 Lys Arg Leu Arg Thr Glu Ser Pro Phe Pro Cys Leu Phe Ala Glu Glu 35 40 45 gcc tac cag aaa ctg gcc agc gag acc ctg gag gag ctg gac tgg tgt 192 Ala Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys 50 55 60 ctg gac cag cta gag acc cta cag acc agg cac tcc gtc agt gag atg 240 Leu Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met 65 70 75 gcc tcc aac aag ttt aaa agg atg ctt aat cgg gag ctc acc cat ctc 288 Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu 80 85 90 95 tct gaa atg agt cgg tct gga aat caa gtg tca gag ttt ata tca aac 336 Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn 100 105 110 aca ttc tta gat aag caa cat gaa gtg gaa att cct tct cca act cag 384 Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln 115 120 125 aag gaa aag gag aaa aag aaa aga cca atg tct cag atc agt gga gtc 432 Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val 130 135 140 aag aaa ttg atg cac agc tct agt ctg act aat tca agt atc cca agg 480 Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg 145 150 155 ttt gga gtt aaa act gaa caa gaa gat gtc ctt gcc aag gaa cta gaa 528 Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu 160 165 170 175 gat gtg aac aaa tgg ggt ctt cat gtt ttc aga ata gca gag ttg tct 576 Asp Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser 180 185 190 ggt aac cgg ccc ttg act gtt atc atg cac acc att ttt cag gaa cgg 624 Gly Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg 195 200 205 gat tta tta aaa aca ttt aaa att cca gta gat act tta att aca tat 672 Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr 210 215 220 ctt atg act ctc gaa gac cat tac cat gct gat gtg gcc tat cac aac 720 Leu Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn 225 230 235 aat atc cat gct gca gat gtt gtc cag tct act cat gtg cta tta tct 768 Asn Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser 240 245 250 255 aca cct gct ttg gag gct gtg ttt aca gat ttg gag att ctt gca gca 816 Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala 260 265 270 att ttt gcc agt gca ata cat gat gta gat cat cct ggt gtg tcc aat 864 Ile Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn 275 280 285 caa ttt ctg atc aat aca aac tct gaa ctt gcc ttg atg tac aat gat 912 Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp 290 295 300 tcc tca gtc tta gag aac cat cat ttg gct gtg ggc ttt aaa ttg ctt 960 Ser Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu 305 310 315 cag gaa gaa aac tgt gac att ttc cag aat ttg acc aaa aaa caa aga 1008 Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg 320 325 330 335 caa tct tta agg aaa atg gtc att gac atc gta ctt gca aca gat atg 1056 Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met 340 345 350 tca aaa cac atg aat cta ctg gct gat ttg aag act atg gtt gaa act 1104 Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr 355 360 365 aag aaa gtg aca agc tct gga gtt ctt ctt ctt gat aat tat tcc gat 1152 Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp 370 375 380 agg att cag gtt ctt cag aat atg gtg cac tgt gca gat ctg agc aac 1200 Arg Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn 385 390 395 cca aca aag cct ctc cag ctg tac cgc cag tgg acg gac cgg ata atg 1248 Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met 400 405 410 415 gag gag ttc ttc cgc caa gga gac cga gag agg gaa cgt ggc atg gag 1296 Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu 420 425 430 ata agc ccc atg tgt gac aag cac aat gct tcc gtg gaa aaa tca cag 1344 Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln 435 440 445 gtg ggc ttc ata gac tat att gtt cat ccc ctc tgg gag aca tgg gca 1392 Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala 450 455 460 gac ctc gtc cac cct gac gcc cag gat att ttg gac act ttg gag gac 1440 Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp 465 470 475 aat cgt gaa tgg tac cag agc aca atc cct cag agc ccc tct cct gca 1488 Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala 480 485 490 495 cct gat gac cca gag gag ggc cgg cag ggt caa act gag aaa ttc cag 1536 Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln 500 505 510 ttt gaa cta act tta gag gaa gat ggt gag tca gac acg gaa aag gac 1584 Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp 515 520 525 agt ggc agt caa gtg gaa gaa gac act agc tgc agt gac tcc aag act 1632 Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr 530 535 540 ctt cgt act caa gac tca gag tct act gaa att ccc ctt gat gaa cag 1680 Leu Arg Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln 545 550 555 gtt gaa gag gag gca gta ggg gaa gaa gag gaa agc caa cct gaa gcc 1728 Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala 560 565 570 575 tgt gtc ata gat gat cgt tct cct gac acg taa cagtgcaaa 1770 Cys Val Ile Asp Asp Arg Ser Pro Asp Thr * 580 585 <210> SEQ ID NO 19 <211> LENGTH: 1684 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (152)...(1675) <400> SEQUENCE: 19 aatatgaagg agcagccctc atgtgccggc accgggcatc cgagcatggc gggaggaggc 60 ctaccagaaa ctggccagcg agaccctgga ggagctggac tggtgtctgg accagctaga 120 gaccctacag accaggcact ccgtcagtga g atg gcc tcc aac aag ttt aaa 172 Met Ala Ser Asn Lys Phe Lys 1 5 agg atg ctt aat cgg gag ctc acc cat ctc tct gaa atg agt cgg tct 220 Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser 10 15 20 gga aat caa gtg tca gag ttt ata tca aac aca ttc tta gat aag caa 268 Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe Leu Asp Lys Gln 25 30 35 cat gaa gtg gaa att cct tct cca act cag aag gaa aag gag aaa aag 316 His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys Glu Lys Lys 40 45 50 55 aaa aga cca atg tct cag atc agt gga gtc aag aaa ttg atg cac agc 364 Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys Leu Met His Ser 60 65 70 tct agt ctg act aat tca agt atc cca agg ttt gga gtt aaa act gaa 412 Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly Val Lys Thr Glu 75 80 85 caa gaa gat gtc ctt gcc aag gaa cta gaa gat gtg aac aaa tgg ggt 460 Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val Asn Lys Trp Gly 90 95 100 ctt cat gtt ttc aga ata gca gag ttg tct ggt aac cgg ccc ttg act 508 Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg Pro Leu Thr 105 110 115 gtt atc atg cac acc att ttt cag gaa cgg gat tta tta aaa aca ttt 556 Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe 120 125 130 135 aaa att cca gta gat act tta att aca tat ctt atg act ctc gaa gac 604 Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met Thr Leu Glu Asp 140 145 150 cat tac cat gct gat gtg gcc tat cac aac aat atc cat gct gca gat 652 His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile His Ala Ala Asp 155 160 165 gtt gtc cag tct act cat gtg cta tta tct aca cct gct ttg gag gct 700 Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Glu Ala 170 175 180 gtg ttt aca gat ttg gag att ctt gca gca att ttt gcc agt gca ata 748 Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ser Ala Ile 185 190 195 cat gat gta gat cat cct ggt gtg tcc aat caa ttt ctg atc aat aca 796 His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr 200 205 210 215 aac tct gaa ctt gcc ttg atg tac aat gat tcc tca gtc tta gag aac 844 Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser Val Leu Glu Asn 220 225 230 cat cat ttg gct gtg ggc ttt aaa ttg ctt cag gaa gaa aac tgt gac 892 His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu Asn Cys Asp 235 240 245 att ttc cag aat ttg acc aaa aaa caa aga caa tct tta agg aaa atg 940 Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu Arg Lys Met 250 255 260 gtc att gac atc gta ctt gca aca gat atg tca aaa cac atg aat cta 988 Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys His Met Asn Leu 265 270 275 ctg gct gat ttg aag act atg gtt gaa act aag aaa gtg aca agc tct 1036 Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser 280 285 290 295 gga gtt ctt ctt ctt gat aat tat tcc gat agg att cag gtt ctt cag 1084 Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Gln 300 305 310 aat atg gtg cac tgt gca gat ctg agc aac cca aca aag cct ctc cag 1132 Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Gln 315 320 325 ctg tac cgc cag tgg acg gac cgg ata atg gag gag ttc ttc cgc caa 1180 Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Arg Gln 330 335 340 gga gac cga gag agg gaa cgt ggc atg gag ata agc ccc atg tgt gac 1228 Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp 345 350 355 aag cac aat gct tcc gtg gaa aaa tca cag gtg ggc ttc ata gac tat 1276 Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr 360 365 370 375 att gtt cat ccc ctc tgg gag aca tgg gca gac ctc gtc cac cct gac 1324 Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp 380 385 390 gcc cag gat att ttg gac act ttg gag gac aat cgt gaa tgg tac cag 1372 Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr Gln 395 400 405 agc aca atc cct cag agc ccc tct cct gca cct gat gac cca gag gag 1420 Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp Pro Glu Glu 410 415 420 ggc cgg cag ggt caa act gag aaa ttc cag ttt gaa cta act tta gag 1468 Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu Thr Leu Glu 425 430 435 gaa gat ggt gag tca gac acg gaa aag gac agt ggc agt caa gtg gaa 1516 Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser Gln Val Glu 440 445 450 455 gaa gac act agc tgc agt gac tcc aag act ctt cgt act caa gac tca 1564 Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Arg Thr Gln Asp Ser 460 465 470 gag tct act gaa att ccc ctt gat gaa cag gtt gaa gag gag gca gta 1612 Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu Glu Glu Ala Val 475 480 485 ggg gaa gaa gag gaa agc caa cct gaa gcc tgt gtc ata gat gat cgt 1660 Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val Ile Asp Asp Arg 490 495 500 tct cct gac acg taa cagtgcaaa 1684 Ser Pro Asp Thr 505 <210> SEQ ID NO 20 <211> LENGTH: 2105 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (75)...(2096) <400> SEQUENCE: 20 ggaattcatc tgtaaaaatc actacatgta acgtaggaga caagaaaaat attaatgaca 60 gaagatctgc gaac atg atg cac gtg aat aat ttt ccc ttt aga agg cat 110 Met Met His Val Asn Asn Phe Pro Phe Arg Arg His 1 5 10 tcc tgg ata tgt ttt gat gtg gac aat ggc aca tct gcg gga cgg agt 158 Ser Trp Ile Cys Phe Asp Val Asp Asn Gly Thr Ser Ala Gly Arg Ser 15 20 25 ccc ttg gat ccc atg acc agc cca gga tcc ggg cta att ctc caa gca 206 Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu Ile Leu Gln Ala 30 35 40 aat ttt gtc cac agt caa cga cgg gag tcc ttc ctg tat cga tcc gac 254 Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp 45 50 55 60 agc gat tat gac ctc tct cca aag tct atg tcc cgg aac tcc tcc att 302 Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg Asn Ser Ser Ile 65 70 75 gcc agt gat ata cac gga gat gac ttg att gtg act cca ttt gct cag 350 Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr Pro Phe Ala Gln 80 85 90 gtc ttg gcc agc ctg cga act gta cga aac aac ttt gct gca tta act 398 Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe Ala Ala Leu Thr 95 100 105 aat tcg caa gat cga gca cct agc aaa aga tca ccc aca tgc aac caa 446 Asn Ser Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro Thr Cys Asn Gln 110 115 120 cca tcc atc aac aaa gcc acc ata aca gag gag gcc tac cag aaa ctg 494 Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala Tyr Gln Lys Leu 125 130 135 140 gcc agc gag acc ctg gag gag ctg gac tgg tgt ctg gac cag cta gag 542 Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu 145 150 155 acc cta cag acc agg cac tcc gtc agt gag atg gcc tcc aac aag ttt 590 Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala Ser Asn Lys Phe 160 165 170 aaa agg atg ctt aat cgg gag ctc acc cat ctc tct gaa atg agt cgg 638 Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg 175 180 185 tct gga aat caa gtg tca gag ttt ata tca aac aca ttc tta gat aag 686 Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe Leu Asp Lys 190 195 200 caa cat gaa gtg gaa att cct tct cca act cag aag gaa aag gag aaa 734 Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys Glu Lys 205 210 215 220 aag aaa aga cca atg tct cag atc agt gga gtc aag aaa ttg atg cac 782 Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys Leu Met His 225 230 235 agc tct agt ctg act aat tca agt atc cca agg ttt gga gtt aaa act 830 Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly Val Lys Thr 240 245 250 gaa caa gaa gat gtc ctt gcc aag gaa cta gaa gat gtg aac aaa tgg 878 Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val Asn Lys Trp 255 260 265 ggt ctt cat gtt ttc aga ata gca gag ttg tct ggt aac cgg ccc ttg 926 Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg Pro Leu 270 275 280 act gtt atc atg cac acc att ttt cag gaa cgg gat tta tta aaa aca 974 Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr 285 290 295 300 ttt aaa att cca gta gat act tta att aca tat ctt atg act ctc gaa 1022 Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met Thr Leu Glu 305 310 315 gac cat tac cat gct gat gtg gcc tat cac aac aat atc cat gct gca 1070 Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile His Ala Ala 320 325 330 gat gtt gtc cag tct act cat gtg cta tta tct aca cct gct ttg gag 1118 Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Glu 335 340 345 gct gtg ttt aca gat ttg gag att ctt gca gca att ttt gcc agt gca 1166 Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ser Ala 350 355 360 ata cat gat gta gat cat cct ggt gtg tcc aat caa ttt ctg atc aat 1214 Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn 365 370 375 380 aca aac tct gaa ctt gcc ttg atg tac aat gat tcc tca gtc tta gag 1262 Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser Val Leu Glu 385 390 395 aac cat cat ttg gct gtg ggc ttt aaa ttg ctt cag gaa gaa aac tgt 1310 Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu Asn Cys 400 405 410 gac att ttc cag aat ttg acc aaa aaa caa aga caa tct tta agg aaa 1358 Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu Arg Lys 415 420 425 atg gtc att gac atc gta ctt gca aca gat atg tca aaa cac atg aat 1406 Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys His Met Asn 430 435 440 cta ctg gct gat ttg aag act atg gtt gaa act aag aaa gtg aca agc 1454 Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser 445 450 455 460 tct gga gtt ctt ctt ctt gat aat tat tcc gat agg att cag gtt ctt 1502 Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu 465 470 475 cag aat atg gtg cac tgt gca gat ctg agc aac cca aca aag cct ctc 1550 Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu 480 485 490 cag ctg tac cgc cag tgg acg gac cgg ata atg gag gag ttc ttc cgc 1598 Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Arg 495 500 505 caa gga gac cga gag agg gaa cgt ggc atg gag ata agc ccc atg tgt 1646 Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys 510 515 520 gac aag cac aat gct tcc gtg gaa aaa tca cag gtg ggc ttc ata gac 1694 Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp 525 530 535 540 tat att gtt cat ccc ctc tgg gag aca tgg gca gac ctc gtc cac cct 1742 Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro 545 550 555 gac gcc cag gat att ttg gac act ttg gag gac aat cgt gaa tgg tac 1790 Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr 560 565 570 cag agc aca atc cct cag agc ccc tct cct gca cct gat gac cca gag 1838 Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp Pro Glu 575 580 585 gag ggc cgg cag ggt caa act gag aaa ttc cag ttt gaa cta act tta 1886 Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu Thr Leu 590 595 600 gag gaa gat ggt gag tca gac acg gaa aag gac agt ggc agt caa gtg 1934 Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser Gln Val 605 610 615 620 gaa gaa gac act agc tgc agt gac tcc aag act ctt cgt act caa gac 1982 Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Arg Thr Gln Asp 625 630 635 tca gag tct act gaa att ccc ctt gat gaa cag gtt gaa gag gag gca 2030 Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu Glu Glu Ala 640 645 650 gta ggg gaa gaa gag gaa agc caa cct gaa gcc tgt gtc ata gat gat 2078 Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val Ile Asp Asp 655 660 665 cgt tct cct gac acg taa cagtgcaaa 2105 Arg Ser Pro Asp Thr 670 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 21 aagactttat taaggctgac 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 22 ttggcttcta tctatctcaa 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 23 ttccacatat aaatgctttt 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 ataattttta tttcaaagga 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 ttaagattct aggcactctg 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 cctctcttta ttagtgcagt 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 taaggcacag ccctgggacc 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 ggttaaagtt aaggcacagc 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 atgagggctg ctccttcata 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 catgctcgga tgcccggtgc 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 tcctcccgcc atgctcggat 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 tattcacgtg catcatgttc 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 tctaaaggga aaattattca 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 aacgtggttc cctcgctcac 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 tcaaaacatg tatgtgccac 20 <210> SEQ ID NO 36 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 36 000 <210> SEQ ID NO 37 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 37 000 <210> SEQ ID NO 38 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 38 000 <210> SEQ ID NO 39 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 39 000 <210> SEQ ID NO 40 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 40 000 <210> SEQ ID NO 41 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 41 000 <210> SEQ ID NO 42 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 42 000 <210> SEQ ID NO 43 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 43 000 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 aaggctgtat ccagggaatt 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 gctaaataaa gtagtctatg 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 agcctcaggg ctaccgagag 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 gctggcccga gagccttcct 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 tctgcctcca tcctggctcg 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 ggtctccacc gcgtaggagg 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 ctggatttct tcaggccggg 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 tgagtccctg gaacgaggag 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 aaaatttgct tggagaatta 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 cactggcaat ggaggagttc 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 atctccgtgt atatcactgg 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 caaagttgtt tcgtacagtt 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 cttgcaaatt agttaatgca 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 ttgctaggtg ctcgatcttg 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 catgggtgat cttttgctag 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 gtggctttgt tgatggatgg 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 cggagtgcct ggtctgtagg 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 tgagctcccg attaagcatc 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 agaatgtgtt tgatataaac 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 tgttgcttat ctaagaatgt 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 ggaatttcca cttcatgttg 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 gttccttggc aaggacatct 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 acaactctgc tattctgaaa 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 tatctactgg aattttaaat 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 atctgcagca tggatattgt 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 ctgtaaacac agcctccaaa 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 tgtattgcac tggcaaaaat 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 catcatgtat tgcactggca 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 72 gtttgtattg atcagaaatt 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 cattttcctt aaagattgtc 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 atgaccattt tccttaaaga 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 tgcaagtacg atgtcaatga 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 tctgaagaac ctgaatccta 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 aacaatatag tctatgaagc 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 78 gtcatcaggt gcaggagagg 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 79 cttgactgcc actgtccttt 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 80 ttgcactgtt acgtgtcagg 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 81 gttgtggcat gtgacatgca 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 82 tggttacgat attcctgagc 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 83 tgtcagctct accaagctga 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 84 aaccccaagt ccaataaact 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 85 gctcatcctc ctcctactgg 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 86 cttgcaagtt actccttaga 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 87 tagaacaaat agtcactttg 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 88 agatgaattc caactctctt 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 89 tgttcgcaga tcttctgtca 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 90 cacatcaaaa catatccagg 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 91 gtatatgggt tcaattccat 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 92 gaatttccac cagtcagcat 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 93 atccttttaa actgttatgg 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 94 gcacggagca ggaggcactt 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 95 aaactgtagg actttgagaa 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 96 cacatcaaaa ctgtaggact 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 97 aagctgatga atagataacc 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 98 taaatgtttc ctgctttagg 20 <210> SEQ ID NO 99 <211> LENGTH: 1430 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (19)...(66) <400> SEQUENCE: 99 gacagaagat ctgcgaac atg atg cac gtg aat aat ttt ccc ttt aga agg 51 Met Met His Val Asn Asn Phe Pro Phe Arg Arg 1 5 10 cat tcc tgg ata tga ggaggcctac cagaaactgg ccagcgagac cctggaggag 106 His Ser Trp Ile * 15 ctggactggt gtctggacca gctagagacc ctacagacca ggcactccgt cagtgagatg 166 gcctccaaca agtttaaaag gatgcttaat cgggagctca cccatctctc tgaaatgagt 226 cggtctggaa atcaagtgtc agagtttata tcaaacacat tcttagataa gcaacatgaa 286 gtggaaattc cttctccaac tcagaaggaa aaggagaaaa agaaaagacc aatgtctcag 346 atcagtggag tcaagaaatt gatgcacagc tctagtctga ctaattcaag tatcccaagg 406 tttggagtta aaactgaaca agaagatgtc cttgccaagg aactagaaga tgtgaacaaa 466 tggggtcttc atgttttcag aatagcagag ttgtctggta accggccctt gactgttatc 526 atgcacacca tttttcagga acgggattta ttaaaaacat ttaaaattcc agtagatact 586 ttaattacat atcttatgac tctcgaagac cattaccatg ctgatgtggc ctatcacaac 646 aatatccatg ctgcagatgt tgtccagtct actcatgtgc tattatctac acctgctttg 706 gaggctgtgt ttacagattt ggagattctt gcagcaattt ttgccagtgc aatacatgat 766 gtagatcatc ctggtgtgtc caatcaattt ctgatcaata caaactctga acttgccttg 826 atgtacaatg attcctcagt cttagagaac catcatttgg ctgtgggctt taaattgctt 886 caggaagaaa actgtgacat tttccagaat ttgaccaaaa aacaaagaca atctttaagg 946 aaaatggtca ttgacatcgt acttgcaaca gatatgtcaa aacacatgaa tctactggct 1006 gatttgaaga ctatggttga aactaagaaa gtgacaagct ctggagttct tcttcttgat 1066 aattattccg ataggattca ggttcttcag aatatggtgc actgtgcaga tctgagcaac 1126 ccaacaaagc ctctccagct gtaccgccag tggacggacc ggataatgga ggagttcttc 1186 cgccaaggag accgagagag ggaacgtggc atggagataa gccccatgtg tgacaagcac 1246 aatgcttccg tggaaaaatc acaggtgggc ttcatagact atattgttca tcccctctgg 1306 gagacatggg cagacctcgt ccaccctgac gcccaggata ttttggacac tttggaggac 1366 aatcgtgaat ggtaccagag cacaatccct cagagcccct ctcctgcacc tgatgaccca 1426 gagg 1430 <210> SEQ ID NO 100 <211> LENGTH: 2025 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(648) <400> SEQUENCE: 100 atg gct cag cag aca agc ccg gac act tta aca gta cct gaa gtg gat 48 Met Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu Val Asp 1 5 10 15 aat ccg cat tgt cca aac ccg tgg ctg aac gaa gac ctt gtg aaa tcc 96 Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val Lys Ser 20 25 30 ttg cga gaa aac ctg ttg cag cat gag aag tcc aag aca gcg agg aaa 144 Leu Arg Glu Asn Leu Leu Gln His Glu Lys Ser Lys Thr Ala Arg Lys 35 40 45 tcg gtt tct ccc aag ctc tct cca gtg atc tct ccg aga aat tcc ccc 192 Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn Ser Pro 50 55 60 agg ctt ctg cgc aga atg ctt ctc agc agc aac atc ccc aaa cag cgg 240 Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys Gln Arg 65 70 75 80 cgt ttc acg gtg gca cat aca tgt ttt gat gtg gac aat ggc aca tct 288 Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly Thr Ser 85 90 95 gcg gga cgg agt ccc ttg gat ccc atg acc agc cca gga tcc ggg cta 336 Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu 100 105 110 att ctc caa gca aat ttt gtc cac agt caa cga cgg gag tcc ttc ctg 384 Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu 115 120 125 tat cga tcc gac agc gat tat gac ctc tct cca aag tct atg tcc cgg 432 Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg 130 135 140 aac tcc tcc att gcc agt gat ata cac gga gat gac ttg att gtg act 480 Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr 145 150 155 160 cca ttt gct cag gtc ttg gcc agt ctg cga act gta cga aac aac ttt 528 Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe 165 170 175 gct gca tta act aat ttg caa gat cga gca cct agc aaa aga tca ccc 576 Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro 180 185 190 atg tgc aac caa cca tcc atc aac aaa gcc acc ata aca ggg ctc tat 624 Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Gly Leu Tyr 195 200 205 aat ggg atc att gca ttt ctc tga ttactaatga gaaggaggcc taccagaaac 678 Asn Gly Ile Ile Ala Phe Leu * 210 215 tggccagcga gaccctggag gagctggact ggtgtctgga ccagctagag accctacaga 738 ccaggcactc cgtcagtgag atggcctcca acaagtttaa aaggatgctt aatcgggagc 798 tcacccatct ctctgaaatg agtcggtctg gaaatcaagt gtcagagttt atatcaaaca 858 cattcttaga taagcaacat gaagtggaaa ttccttctcc aactcagaag gaaaaggaga 918 aaaagaaaag accaatgtct cagatcagtg gagtcaagaa attgatgcac agctctagtc 978 tgactaattc aagtatccca aggtttggag ttaaaactga acaagaagat gtccttgcca 1038 aggaactaga agatgtgaac aaatggggtc ttcatgtttt cagaatagca gagttgtctg 1098 gtaaccggcc cttgactgtt atcatgcaca ccatttttca ggaacgggat ttattaaaaa 1158 catttaaaat tccagtagat actttaatta catatcttat gactctcgaa gaccattacc 1218 atgctgatgt ggcctatcac aacaatatcc atgctgcaga tgttgtccag tctactcatg 1278 tgctattatc tacacctgct ttggaggctg tgtttacaga tttggagatt cttgcagcaa 1338 tttttgccag tgcaatacat gatgtagatc atcctggtgt gtccaatcaa tttctgatca 1398 atacaaactc tgaacttgcc ttgatgtaca atgattcctc agtcttagag aaccatcatt 1458 tggctgtggg ctttaaattg cttcaggaag aaaactgtga cattttccag aatttgacca 1518 aaaaacaaag acaatcttta aggaaaatgg tcattgacat cgtacttgca acagatatgt 1578 caaaacacat gaatctactg gctgatttga agactatggt tgaaactaag aaagtgacaa 1638 gctctggagt tcttcttctt gataattatt ccgataggat tcaggttctt cagaatatgg 1698 tgcactgtgc agatctgagc aacccaacaa agcctctcca gctgtaccgc cagtggacgg 1758 accggataat ggaggagttc ttccgccaag gagaccgaga gagggaacgt ggcatggaga 1818 taagccccat gtgtgacaag cacaatgctt ccgtggaaaa atcacaggtg ggcttcatag 1878 actatattgt tcatcccctc tgggagacat gggcagacct cgtccaccct gacgcccagg 1938 atattttgga cactttggag gacaatcgtg aatggtacca gagcacaatc cctcagagcc 1998 cctctcctgc acctgatgac ccagagg 2025 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding phosphodiesterase 4D, wherein said compound specifically hybridizes with said nucleic acid molecule encoding phosphodiesterase 4D and inhibits the expression of phosphodiesterase 4D.
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 4. The compound of claim 3 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 5. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 6. The compound of claim 5 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 8. The compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound 8 to 80 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding phosphodiesterase 4D.
 11. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 12. The composition of claim 11 further comprising a colloidal dispersion system.
 13. The composition of claim 11 wherein the compound is an antisense oligonucleotide.
 14. A method of inhibiting the expression of phosphodiesterase 4D in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of phosphodiesterase 4D is inhibited.
 15. A method of treating an animal having a disease or condition associated with phosphodiesterase 4D comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of phosphodiesterase 4D is inhibited.
 16. The method of claim 15 wherein the disease or condition is cancer.
 17. The method of claim 16 wherein the disease or condition is cardiovascular disease.
 18. The method of claim 15 wherein the disease or condition is inflammation.
 19. The compound of claim 1 targeted to a nucleic acid molecule encoding phosphodiesterase 4D, wherein said compound specifically hybridizes with and differentially inhibits the expression of one of the variants of phosphodiesterase 4D relative to the remaining variants of phosphodiesterase 4D.
 20. The compound of claim 19 targeted to a nucleic acid molecule encoding phosphodiesterase 4D, wherein said compound hybridizes with and specifically inhibits the expression of a variant of phosphodiesterase 4D, wherein said variant is selected from the group consisting of: PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4DH1, PDE4DH2, PDE4DN1, PDE4DN2 and PDE4DN3. 